The direct FAC-MS method is characterized by an online coupling of mass spec-trometry with the FAC cartridge . This is usually enabled with electrospray as an ionization method especially when monitoring drug-like molecules, although MALDI has also proven useful [15, 16]. MS detection might seem less significant when making binding measurements on single ligands; however it is very important when making these measurements that the void volume be accurately determined. This can be approximated by co-infusing one or more compounds that are not expected to bind to the stationary phase. For example, our laboratory uses a collection of peptides, oligosaccharides or low concentration buffer components (e.g. nonbuffering Tris concentration) to make V0 measurements, and therefore MS detection is essential. Guiochon has demonstrated that weaker ligands are particularly prone to inaccuracies in the estimation of V0. MS detection enables a more accurate assessment of V0 through multiple sampling.
One of the most useful adaptations of the direct FAC-MS method for quantitat-ing a binding event is referred to as the staircase method (Fig. 6.5). In this method, successively higher concentrations of test ligand are infused and the breakthrough volumes induced by the concentration increments are determined [9, 10]. As with all thorough studies of a binding event, these measurements are best conducted over a wide concentration range so that the binding isotherm is adequately sampled. This staircase method avoids long washing steps between injections; such long washes can be problematic for affinity columns that have a short lifetime. The concentration-series data can be linearized very simply according to Eq. (2) :
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