Development of generic methods for the analysis and purification of polar compounds by high performance liquid chromatography

Generic methods were developed using different columns for analysis and purification of hydrophilic compounds by hydrophilic interaction chromatography (HILIC). Mobile phases were investigated in detail, and across each column chemistry tested (BEH Amide, Atlantis bare silica, ZIC-HILIC and Cogent Hydride), salt-buffered mobile phase offered good to excellent peak shape for acids, bases and neutral solutes with a range of hydrophilicities. Additionally, cation exchange occurred on the bare silica column even when rubidium nitrate was added to the mobile phase, which should block all cation exchange sites. Measurement of mobile phase pH in hydroorganic solvent (ACN-water mixture with buffer) better represented the environment solutes experience on column than fully-aqueous pH measurement. The performance of HILIC with Charged Aerosol Detection (CAD) was evaluated with a hydrophilic acid, a hydrophobic base and a hydrophilic neutral solute; limits of detection and quantitation were 1-3 ng and 5-9 ng on column, respectively. This compared favourably to literature values for other universal detectors. HILIC-CAD was further investigated by flow injection analysis (FIA) using 29 solutes containing acids, bases and neutrals. HILIC and CAD had excellent compatibility: peak areas were double compared to reversed-phase conditions, response was reasonably uniform for 21 non-volatile solutes considering the solutes’ diversity. HILIC-CAD was viable for retention and detection of highly hydrophilic species without chromophores: salts, sugars and amino acids. Salts travelled down the column as independent cations and anions. Resolution of sugars and amino acids was challenging and was incomplete due to project time constraints. Generic methods were developed on an analytical system in the labs of the industrial collaborator and applied to purifications on wide-bore columns at scaled-up flow rates (21mm id, 20mL/min prep vs. 4.6mm id 1 mL / min analytical analytical). A standard prep system was capable of usable productivity using HILIC with 1mL injections (22 mg of crude purified per hour) and use of At-Column Dilution enhanced this around 10-fold with scope for 4mL injections (223 mg of crude purified per hour).