Evaluation of liquid chromatographic behavior of restricted-access media precolumns in the course of direct injection of large volumes of plasma samples in column-switching systems.
The chromatographic behavior of an alkyl-diol silica (ADS, 25 x 4 mm I.D.) and a semipermeable surface (SPS, 10 x 10 mm I.D.) supports two types of restricted-access media (RAM), which served as precolumns in column-switching systems for direct injection of large volumes of plasma samples (500 microl), was studied with regard to peak performance, retention and column back pressure. The adsorption of matrix proteins both on sealings (porous frits and sieves) and packings was also examined. Columns of ADS and SPS were unchanged after the injection of 10-20 ml human plasma under normal working conditions. Even when changes occurred on the precolumns (>50 ml of plasma in total), it was still possible to regenerate the column performance by replacing the column sieves, or by washing and removing columns from the system for a period, since the changes were more related to the blockage of sealings and/or the adsorption of proteins on the hydrophilic surfaces. Proteins could eventually be unspecifically adsorbed on the hydrophobic ligand of the support. It was found on one ADS column that the retention decreased by 20% and the pressure increased 30 bar after an intensive loading of 75 ml plasma (injection volume, 500 microl) without reconditioning procedure. Studies showed that the column sealings played the most important role for the lifetime of RAM columns. For ADS columns, using sieves without polytetrafluoroethylene (PTFE) nets were the best. No significant difference in column life span between SPS and ADS was found.
Yu
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Analytical Pharmaceutical Chemistry, Uppsala University Biomedical Centre, Sweden.
Westerlund
D
D
Boos
K S
KS
D000327:Adsorption; D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D010539:Permeability; D011312:Pressure; D011506:Proteins / Q000737:chemistry; D012822:Silicon Dioxide; D013499:Surface Properties
D011506:Proteins; D012822:Silicon Dioxide