Stationary phase column operation evaluation

Karlsson and Hermansson [30] used chemometrics for optimization of chiral separation of omeprazole and one of its metabolites on immobilized al-acid glycoprotein. Plasma was centrifuged at 2500 rpm and a portion (20-50 ji ) was injected into a 5-/rm Chiral-AGP column (10 cm x 4 mm) with al-acid glycoprotein immobilized to silica as a chiral stationary phase and acetonitrile-phosphate buffer of pH 5.7-7.2 as mobile phase (1 ml/min). Detection of omeprazole and its main metabolite, hydroxy-lated omeprazole, was performed at 302 nm. A statistical model was developed for the optimization of the operational parameters. The experimental data were evaluated with multivariate analyses column temperature and acetonitrile concentration were the most important variables for the enantioseparations. Complete enantiomeric separation for omeprazole and hydoxylated omeprazole was obtained within 15 min. 

From the view point of the assessment, the quality of an HPLC separation in response to changes in different system variables, such as the stationary phase particle diameter, the column configuration, the flow rate, or mobile phase composition, or alternatively, changes in a solute variable such as the molecular size, net charge, charge anisotropy, or hydrophobic cluster distribution of a protein, can be based on evaluation of the system peak capacity (PC) in the analytical modes of HPLC separations and the system productivity (Peff) parameters in terms of bioactive mass recovered throughput per unit time at a specified purity level and operational cost structure. The system peak capacity PC depends on the relative selectivity and the bandwidth, and can be defined as... 

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