Restrictedaccess Media Online Fundamentals

Another technique that allows for the direct injection of plasma or serum online with the chromatographic system uses an analytical column containing particles referred to as restricted-access media (RAM); traditional laminar flow liquid chromatography is employed. These RAM particles are designed to prevent or restrict large macromolecules from accessing the inner adsorption sites of the bonded phase. Commercially available RAM columns, all silica based, include internal surface reverse phase (ISRP), semipermeable surface (SPS), and a hydrophobic shielded phase named HisepTM (Supelco, Bellefonte, Pennsylvania). The most popular column variety used in bioanalysis is the ISRP column; the internal surface is covered with a bonded reversephase material and the external surface is covered with a nonadsorptive but hydrophilic material. This dual-phase column permits effective separation of the analyte of interest from macromolecules in the sample matrix; drugs and other small molecules enter the pores of the hydrophobic reverse phase to partition and be retained, while proteins and larger matrix components are excluded. Essentially, a combination of size exclusion chromatography and partition chromatography is observed. When serum or plasma is injected onto a RAM column, the proteins are excluded by the outer, hydrophilic layer and pass through to waste. Note that conventional polymers (e.g., Oasis HLB) are subject to protein adsorption from nonspecific binding; column lifetimes are reduced tenfold compared with RAM columns [116].

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