Improvement in the resolution of o-phthalaldehyde derivatized amino acids by applying gradient steepness optimization to five reversed-phase columns of different lengths and particle sizes.
Twenty-two o-phthalaldehyde (OPA) derivatized amino acids were separated on five different reversed-phase, octadecyl columns. Each column was packed with the same type of spherical silica on which matched bonding chemistry had been performed. Columns of 250, 150 or 50 mm x 4.5 mm I.D. were packed with 5 microns particles, and columns of 100 or 50 mm x 4.5 mm I.D. were packed with 3 microns particles. Snyder's linear solvent strength gradient optimization method was used to determine the optimum gradient steepness by maximizing the resolution of four pairs of adjacent OPA-derivatized amino acids. An asymptotic dependence of improvement in resolution with increase in gradient time was obtained for each pair of compounds on each column. For short (50 mm) columns, resolution of very closely eluting compounds required the use of gradient steepness parameters as low as 0.05 and 0.02, due to the low efficiencies intrinsic to these short column lengths.
Department of Chemistry, University of Connecticut, Storrs 06268.
M
M
Eslami
J D
JD
Stuart
K H
KH
Chen
D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D010316:Particle Size; D012997:Solvents; D009764:o-Phthalaldehyde
D000596:Amino Acids; D012997:Solvents; D009764:o-Phthalaldehyde