Field-Flow Fractionation Analysis of Complex Biological Samples

Normal analytical methods have difficulties when analysing complex samples containing particles of different size. In the 1960s, a new analytical technique was developed, which was able to overcome those difficulties. This new, Field-Flow Fractionation (FFF) technique has been primarily used in the separation of large particles such as macromolecules and colloids. The development and improvement of the FFF technique led to the coupling of the technique with other specific and sensitive analytical methods which resulted in the FFF technique becoming very useful in isolation, separation and analysis of various complex samples, such as powders, emulsions, colloids, geological sediments, biopolymers, complex proteins, polysaccharides, synthetic polymers, and many others. The separation field in the FFF technique is a thin, empty flow chamber called a channel. The structure of the ribbonlike channel with view of the parabolic flow can be seen in Fig. 1. Separation is achieved by the interaction of sample components with an externally generated field, which is applied perpendicularly to the direction of the mobile flow inside the channel. Sample components, which differ in molar mass, size or other properties are pushed by the applied perpendicular field into different velocity regions within the parabolic flow profile of the mobile phase across the channel. The flow has different velocity depending on the position within the channel; the velocity at the walls is zero and it increases towards the centre of the channel. Samples are carried downstream through the channel at different velocities and exit the channel after different retention times. The relative distribution of samples in the parabolic flow determines the separation characteristics. Different operating modes have different types of distributions. The most frequently used mechanisms of FFF separation are listed in Fig. 2. Based on the characteristics of analysed particles and applied outer perpendicular field, FFF techniques can be divided into Sedimentation, Flow, Thermal, Electrical and Magnetic techniques. The FFF techniques versatility, advantages, disadvantages and wide-range application of every specified FFF technique are listed in this review article. Coupling of different FFF techniques with simple or more comprehensive detectors, such as UV/VIS, MALS or mass spectrometer (see Fig. 6) has established the FFF as an efficient and versatile technique. The successful implementation of the FFF technique in the analysis of complex biological samples would influence the future development and direction of the technique's development