Commercial flow field-flow fractionation (FlFFF) employs macro-scale, flat-type channels. The idea of hollow-fiber (HF) membranes as tubular, micro-column channels for FlFFF (HF FlFFF or, more shortly, HF5) dates back to 1974, with fundamentals on HF5 given in the late 1980s, and outstanding applications reported only over the last 15 years.Compared to flat-channel FlFFF, the key aspect of HF5 lies in the downscaling of the fractionation channel. This implies low-cost, possible disposable usage, and low volume of the channel that allows on-line coupling with highly sensitive detection and characterization techniques. The use of coupled techniques enhances the analysis of macromolecules and micron-sized particles such as intact proteins and whole cells. In this chapter we first report a few basics on HF5 theory and instrumentation. We then focus on technical and methodological developments that have made HF5 reach a performance normally achieved by flat-channel FlFFF. We finally focus on the enhancements obtained by coupling HF5 with powerful methods for detection and characterization of intact proteins and whole cells such as multi-angle light scattering (MALS), time-of-flight (TOF) mass spectrometry (MS), chemiluminescence (CL), and UV/Vis turbidity diode-array detection (DAD).
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