Asymmetrical flow field-flow fractionation (AF4) is one of the most widely used FFF techniques for separating polymers, nanoparticles, and proteins. Implemented in 1987, AF4 uses a semi-permeable wall and has an external field that is a perpendicular crossflow of the same fluid that is used in the axial flow stream. This perpendicular crossflow constantly flows through the channel and exits through a semi-permeable wall which retains particles at the accumulation wall. By using an inverse axial flow, or focusing flow of the same carrier fluid, the sample of interest can align at the beginning of the channel. Once the focusing flow is turned off, the particles in the sample can then flow through the channel and be separated by their varying rates of diffusion. 

When an analyte is placed in a thermal gradient it will typically diffuse towards the colder region of the gradient, this is known as thermal diffusion or thermophoresis. The magnitude of this force is dependent on the strength of the thermal field and composition of the analyte and carrier fluid(s). This technique is generally used to separate analytes which undergo Brownian motion, however, it may be applied to particles or polymers of larger sizes. A major limitation, and area of research for our group, is the lack of a complete theory on thermophoresis in liquid phase. Thus the rate of thermal diffusion must be determined experimentally and then separation may be optimized or exploited to yield compositional information.

In SdFFF the channel is wrapped around a centrifuge and rotated at high speeds where the acceleration force is perpendicular to the flow. This technique has the highest resolution of the FFF family of techniques. This is rooted in the relationship between the field’s force and the size of the analyte, which is diameter to the third power. Due to the very large difference in retention time of analytes a programmed field decay is often incorporated to reduce run time. 

As with column chromatography, we employ a variety of online detection systems to provide detailed information about analytes we separate. A flow-through ultra-violet (UV) spectrophotometer is used to detect and quantify UV absorbing materials. In addition to UV detection, multi-angle light scattering (MALS) is leveraged to provide important information for both polymers and biological analytes. From MALS we may determine the second virial coefficient (A2), the root-mean square radius (RMS), and a weight-averaged molecular weight (Mw) of an analyte. When combined with a FFF technique, we may determine this information for sample fractions which allows us to infer more information than may be obtained in batch mode. To compliment MALS we also incorporate dynamic light scattering (DLS), which is also known as photon correlation spectroscopy and quasi-elastic light scattering. DLS  provides us with the translation diffusion of the fraction which can be used to calculate a hydrodynamic radius through the Stokes-Einstein relation. An orthogonal method for determining the hydrodynamic radius is viscometry. Our online detectors may be used to determine the intrinsic viscosity which can then be used to calculate the analyte’s spherical radius via the Einstein-Simha relation. With both hydrodynamic radius and RMS radius we may extract morphological information about fractionated analytes. To determine the concentration of species which do not absorb UV radiation, we use a differential refractometer.

In many cases online detection systems may not provide enough detailed information about an analyte and are subjected to further analysis. The Colorado School of Mines provides a vast array of instrumentation for us to utilize. These include, but are not limited to, MALDI-TOF-MS, electron microscopes, rheometers, and x-ray photoelectron spectroscopy (XPS).