24


May/June 2011


Use of Fortis HILIC stationary phases


Hydrophilic interaction liquid chromatography or HILIC is a technique that has been around for quite some time, based somewhere between reversed-phase (RP) and normalphase (NP) chromatography.


Understanding HILIC


A highly polar stationary phase, often bare silica, is used with largely a non-polar mobile phase system (water acting as a very strong solvent in HILIC) to provide retention of polar or hydrophilic molecules.


The mechanisms of HILIC are still being fully explored but it is quite well


Figure 1. Water layer at the stationary phase surface


accepted now that the analyte partitions between the mobile phase and a water enriched layer at the surface of the


stationary phase. The surface of the stationary phase when underivatized forms this layer with water due to the exposed silanols. Also thought to be part of the retention mechanism is ion-exchange and hydrogen bonding dependant upon the analyte in question.


For example, basic analytes with positive charge will partition into the water layer at the surface, but will also have a cation exchange mechanism with the silanols at the phase surface.


Method Development Adjustment of the retention profile of hydrophilic compounds is made by several potential changes; adjustment of the organic: aqueous eluent, both ratio and organic solvent choice, concentration and type of buffer used, and the pH of the mobile phase. Generally acetonitrile is the common choice for organic, with between 2% and 40% water added in order to elute the compounds in a suitable time frame.


The steep curve of retention (figure 2) is a common theme in HILIC, where at a given point the organic:aqueous ratio is such that the compound suddenly exhibits strong retention where previously little was achieved. Figure 3.


Advantages of HILIC HILIC chromatography provides retention of highly polar hydrophilic analytes that are difficult to retain on RP methods using simple combinations of mobile phase and stationary


Figure 3. Role of Water as modifier in HILIC


Hydrophilic retention Most HILIC applications tend to centre around the analysis of small hydrophilic molecules such as carbohydrates, nucleosides and nucleotides, amino acids and organic acids. However use has been found with diverse molecules such as the application of melamine, figure 4, when there was a worldwide baby milk scare in recent years.


Drugs of abuse, sugars, environmental


Figure 2. Role of organic modifier in HILIC


phase. Many RP options have been tried over the years; polar-endcapping and polar-embedded stationary phases, ion pair reagents and derivatization can all work to a greater or lesser extent.


However all of them have drawbacks either in low retention, stationary phase ”bleed” or lack of compatibility with detection techniques such as mass spectrometry (MS). HILIC provides a distinct advantage in this link up with MS, in that sensitivity is increased


dramatically due to the high organic


concentration and this eases the ionization process during analyte infusion.


Column: p/n:


Fortis HILIC 100x2.1mm 3µ FHI-020503


Mobile Phase: 90:10 ACN : 20mM NH4OAc Flow: Temp:


0.2ml/min 20°C


Wavelength: 210nm


herbicides/pesticides, small peptides and proteins are all


applicable to the mechanisms that HILIC offers. The range is potentially limitless for small hydrophilic molecules.


Figure 5. highlights the ability of Fortis™ HILIC to separate and resolve a mixture (>150 compounds) of peptides, organic acids, sugars, phosphosugars and amino acids within a complex metabolite sample. The Fortis HILIC column is compared with another popular HILIC stationary phase, and exhibits better peak shapes, less co-elution, full baseline resolution for species such as Leucine and Isoleucine. The amine, Spermidine proves difficult to elute with the ZICHILIC stationary phase under these gradient conditions, however the Fortis HILIC column provides good recovery and sensitivity.


UHPLC HILIC


UHPLC is the major talking point at this moment in time for most people in method development and those in chromatography with time or sample limitations. 1.7µm Fortis columns are available with two choices for UHPLC separations. Fortis HILIC an underivatized bare-silica stationary phase, and Fortis HILIC DIOL; a bonded DIOL phase chemistry to aid in the retention and resolution of steroids, proteins and metabolites especially.


The theory of basic analytes causing peak shape problems due to silanol activity is the norm in reversed phase chromatography, however in HILIC the current thought is that vastly superior peak shapes are achieved for


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