SHARC™ HPLC columns are the latest innovation from SIELC Technologies, the inventors of Primesep®. SHARC columns are the first commercially available columns with separation based primarily on hydrogen bonding. SHARC stands for Specific Hydrogen-bond Adsorption Resolution Chromatography.
Hydrogen bonding is an interaction between hydrogen atom bound to electronegative atoms in a molecule, such as oxygen, nitrogen, fluorine. This is typically a weak interaction, especially when separation is performed in aqueous solutions. Liquid chromatography techniques evolved as tool for separation of different molecules based on their physico-chemical properties. Most common techniques of the separation are:
Columns and stationary phases based on these techniques never perform purely with one type of interaction. Hydrogen bonding is omnipresent in every one of these techniques with minor contribution to retention and selectivity. However, in some cases, especially in normal phase chromatography, the contribution of hydrogen bonding can be significant. SHARC 1 is the first column specifically design to perform a separation based entirely on the interaction of the molecules capable providing hydrogen atom (donor) or attract hydrogen atom (acceptor) to the stationary phase with special properties.
SHARC 1 column operation conditions are unique. A mixture of acetonitrile (MeCN), a weak solvent, and methanol (MeOH), a strong solvent, are used as the mobile phase. Pure MeCN has very insignificant amount of hydrogen bonding with the SHARC stationary phase, while MeOH interacts strongly with SHARC stationary phase, which reduces the retention of analytes based on it’s capacity to hydrogen bond. By changing ratio of MeCN/MeOH the optimum retention profile can be obtained for many types of molecules with high selectivity, peak shape, efficiency, and speed.
|Surface interaction within the column with hydrogen bonding capable analytes with acetonitrile as the mobile phase.||Surface interaction within the column with hydrogen bonding capable analytes with methanol as the mobile phase.|
Hydrogen bonding energy is usually in range of 30 kJ/mol or less and depends strongly on the nature of the functional groups and their orientation within the molecule. This energy difference is the basis for selectivity of the interaction between the stationary phase and the molecule of different structure and chemical characteristics.
A given molecule can retain on the stationary phase with more than one hydrogen bond, while also performing as a donor or acceptor of a hydrogen atom.
Presence of a polarized hydrogen atom is not always enough to observe retention by hydrogen bond mechanism. Sometimes a compound can form intramolecular interactions, as oppose to intermolecular hydrogen bonding, and does not participate in stationary phase interaction.
SHARC 1 column is a hydrogen acceptor type stationary phase showing higher retention characteristics toward molecules with higher number of polar X-H bonds such as alcohols, amines, acids, amides, phenols etc.
Hydrogen-bonding interaction offers unique selectivity based on number of “interaction points” available for hydrogen bonding. One of the useful characteristics to determine retention patterns in hydrogen-bonding mode is the molecular polar surface area (PSA). This calculated parameter is usually used for prediction of drug transport properties, but we successfully applied it to hydrogen-bonding interactions. Polar surface area is defined as a sum of surfaces of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule. Since those polar atoms can participate in hydrogen-bonding interaction, estimation of elution order can often be made based on PSA. While PSA is a good indicator of elution time, it must be noted that polar surface area does not account for the accessibility of hydrogen-interaction sites. Not every polar surface participates in intermolecular hydrogen interactions with the stationary phase.
Proximity of “interaction points” to each other within one molecule also needs to be considered since molecules can form an intramolecular hydrogen-bonding, which competes with intermolecular interaction between analyte and stationary phase. This reduces retention time in hydrogen-bonding mode. Such structural factors provides unique selectivity among similarly structural (isomers, homologs, degradation products, precursors) molecules.
Since SHARC 1 column is a mixed-mode column, pKa is another useful parameter in method development for these columns. SHARC columns operate in non-aqueous mobile phase, but some effect of charge interaction of stationary phase and ionizable molecules still exists and contributes to the retention profile.