Characterization of protein aggregates and other biological systems using FFF and SPLITT techniques

HE CHARACTERIZATION OFany sample, especially biological samples, requires a separation step in order to discriminate the various species and contaminants contained within the sample. The most common separation methods are elution based, usually involving chromatographic columns. The benefits of such systems are that the well-characterized fractions are physically separated so that the individual species are quantitated directly and can be collected for further analysis by microscopy or nuclear magnetic resonance (NMR), for example. A more convenient setup may direct the fractions to an on-line detection system, such as an MS, MALS (multiangle light scattering), or viscometric detector. These standalone detectors are currently available with sufficient sophistication so that multidimensional information can be generated from the single analysis. The last advantage of elution-based methods is that automation of the analysis is easy to implement. Field-flow fractionation (FFF) is an alternate member of the elution-based family of methods, 1 “alternate” meaning that no subordination in performance, utility, or importance is intended. FFF offers all of the advantages mentioned above for elution-based methods. Additionally, FFF serves to complement the current chromatographic methods, providing high-resolution separation of high-MW species. In many situations, FFF can be used where chromatographic methods fail and can also provide superior results for sample types, which are problematic for chromatographic methods. In part, this is due to the open-channel structure used for FFF (which deviates from the packed column structure used in chromatography). While FFF provides an analytical-scale, high-resolution separation in an open-channel format, the open-channel equivalent for larger-scale separation is termed SPLITT for split flow thin cell fractionation. 2 The advantages of the open channel format for FFF and SPLITT systems are listed in Table 1. The FFF system and its application to biological samples is described below, followed by a description of the SPLITT system and its applications. Materials and methods FFF channel structure