Evaluation of chromatographic parameters in supercritical fluid chromatography of amino acids as model polar analytes...

Chromatographic parameters have been evaluated regarding the supercritical fluid chromatography of amino acids as model compounds for polar and chargeable substances of biochemical and biopharmaceutical interest. Their inherent hydrophilicity requires suitable chromatographic conditions regarding stationary and mobile phase to obtain suitable solubility and peak performance. Ten stationary phases differing in polarity and type of ligands including neutral and charged ones were investigated regarding selectivity, peak performance and complementarity/orthogonality. The mobile phase composition regarding solvent and type of buffer additives by varying the degree of hydrophobicity of the acids and bases and degree of substitution in case of the basic amines. Addition of water to the methanol solvent was furthermore found crucial for obtaining a high peak performance. Some complementary selectivity could be obtained depending on a choice of column. Temperature and pressure variations basically only influenced retention with no selectivity changes. Suitable conditions obtained for the amino acids were then successfully applied to oligo- and polypeptides enabling a separation of 4 polypeptides and differing in only one of the 39 amino acids, thereby mimicking analysis of biotransformation reaction products. The utilization of water as mobile phase additive and sulfonic acids as buffer agents could serve as a generic methodology for polar and charged compounds such as amino containing biomolecules.