Procedures for direct injections of untreated blood plasma into liquid chromatographic columns with emphasis on a...

The stability of reversed-phase liquid chromatographic systems when untreated blood plasma samples are applied was studied with respect to retention, peak efficiency and column back-pressure. The influence of the content of organic solvents in the eluent, the flow-rate and the particle diameter of the support was investigated. Many of the column-deteriorating effects seem to be due to denaturation of the plasma proteins and the kinetics of this process. The stability is increased by decreasing the content of organic modifier in the eluent. The flow-rate should be moderate, as both high and low flow-rates give decreased stability. The stability increases with increasing particle size of the solid phase. A small pre-column is used (1) as a guard column and (2) to effect a pre-separation of the solute from plasma matrix components by a pre-column venting technique. The pre-column venting technique considerably increases the lifetime of the system and especially the separation column. Under optimal conditions hundreds of injections (10 microliters of plasma) can be performed without loss of stability.