In cases where the separation and fractionation of nanomaterial is not possible using a column with a stationary phase, such as when the nanomaterial may interact with the column packing material and render it unstable under high shear force. Field flow fractionation (FFF) technique can be much helpful. FFF, first introduced by J. Calvin Giddings in 1966 [1, 2], is a family of flow-based separation and characterization techniques developed specifically for macromolecules, colloidal particles, supramolecular assemblies, cells and etc. FFF is featured by the use of an external field applied perpendicular to the direction of sample flow through a thin, elongated flow chamber (channel). A laminar parabolic flow profile develops in the thin channel with a nearly zero flow velocity at channel wall and maximum at the center of channel. The external perpendicular field provides sufficient driving force to allow nanoparticle stay in different parts of flow profile (with non-uniform velocity profile) and therefore induce the separation. The field strength is a most important factor due to its strong effect on the resolution and separation time. Generally, higher field strength is required for lower mass and smaller nanoparticles.
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With its versatility and programmability, FFF is currently widely used in nanomaterial separation and characterization. One advantage in the FFF technique is that there is no stationary phase in the separation - the sample injected comes out intact with minimum loss of material due to non-specific binding. This feature is particularly useful for less stable nanoparticles such as liposomes, or for polymer or protein coated metal nanoparticles that would otherwise interfere with the performance of a traditional GPC column.
Depend upon the types of separation fields, it gives rise to flow (FFFF), sedimentation (sdFFF), thermal (ThFFF), electrical, magnetic acoustic, dielectrophoretic field flow fractionation. Each type of FFF separates materials by a unique set of properties. For example, FFFF separate analytes based solely on difference in their size. In sedimentation FFF, the buoyant mass of analytes contributes to the separation. While in Thermal FFF, compositional characteristic that determine analytes' thermophoretic mobility affects the separation. Of these techniques, FFFF, sdFFF and ThFFF are commercial available and the first two are commonly used for nanomaterial separation and characterization. The theories of FFF techniques will not be a focus here since it was already extensively discussed in other chapters. In this chapter, we will mainly discuss the applications of Flow FFF in nanomaterial separation and characterization and some applications of SdFFF.
In a flow FFF, a second flow stream (so called cross flow) applies perpendicularly to the primary channel flow and permeates through the channel wall to act as a separation field, leading to the separation of materials. Flow FFF includes two different channel designs, symmetric and asymmetric flow. In general, asymmetric FFFF has a better resolution than symmetric FFF. There are two operation modes in FFFF, normal mode and steric/hyperlayer mode. The elution order of analytes is determined by the operating mode. Only the normal mode, also called Brownian mode, is applicable to nanomaterial analytes or macromolecules less than ~1 Âµm. In normal mode, the small particles or macromolecules tend to accumulate in the region of faster stream of parabolic flow profile due to Brownian motion and therefore lead to small particles eluting out earlier than large ones. This is in contrast with Steric/hyperlayer mode where steric inversion effect occurs when Brownian motion of particle becomes negligible with the increasing size (~ 1 Âµm -100 Âµm) of particles and lead to the inversion of elution sequence. Under steric/hyperlayer mode, the injected particles are initially forced to move towards accumulation wall under cross flow field. The small particles approach more closely to the accumulation wall than those large ones and thus retain in the channel longer than large particles. The elution order is therefore from largest to smallest. Also when high velocity flow applies, the particles are subjected to a lifting force which drives larger particle away from accumulation wall and elute out before smaller particles. Flow FFF has been considered a most universal FFF technique because it can fractionate a wide range of nanomaterials, including polymers, lipid-based liposome and emulsion, metallic and semiconductive colloidal nanoparticles, biomolecules such as oligonucleotides, protein, antibody, etc. It is applicable to broad size range with a lower limit determined by the molecular weight cut off of the partition membrane and an upper limit of 100 Âµm.
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The separation of sdFFF is achieved by imposing a gravitational field perpendicular onto nanomaterial analytes entrained in a laminar flow. The applicable size range for SdFFF is ~ 20 nm- 100 Âµm.
The SEC is a most common technique for polymer analysis. FFFF, in conjunction with MALLS, DLS and RI detectors, also provides invaluable information regarding mass, polydispersity, conformation, branching, particle size and size distribution. The most recent progress on the application of FFF techniques in polymer characterization has been covered in two comprehensive reviews [3, 4]. The big advantage of FFFF over its counterpart technique SEC is FFFF offers a minimum shear force on the analyzed polymers without compromising resolution. This feature was demonstrated in Yohannes et al.  work in which poly(N-isopropylacrylamide) was analyzed using both FFFF and SEC. The result show the higher molar mass, where the average molar mass obtained by SEC was much lower than that obtained by FFFF, indicative of shear degradation of polymer in SEC column. FFF also offers flexibility in separation of polymer regardless of pH or ion strength. For example, polyamidoamine (PAMAM) dendrimer consists of an ethylenediamine core and amine surface is weakly charged or uncharged under higher or neutral pH and adsorb on to the neutral packing SEC column, leading to a poor separation. FFFF has successfully provided size based separation of the PAMAM denderimer at a broad range of pH conditions . The size increase caused by the binding of BSA to dendrimer was also detected by FFFF due to its gentle fractionation nature. Polystyrene latex beads (NIST traceable size standards) are very often used as a narrow standard to check the performance of FFFF instrument or to verify the theoretical principles.
Flow FFF has been used extensively in the study of the size, size distribution, drug loading and stability of liposome or lipid based vesicles. Its gentle separation force prevent liposomes or lipid vesicles from degrading, aggregating, resulting in accurate size measurement [7-10].
Flow FFF was also applied for the length separation of high aspect ratio carbon nanotubes [11-14]. Carbon nanotubes are rolled up seamless cylinders of grapheme sheets, exhibiting unparalleled physical, mechanical, and chemical properties which have attracted tremendous interest since it was being discovered in 1991. The length, chirality surface modification determine the solubility, conductivity, mechanical properties of carbon nanotubes, eventually affect their applications. The length separation of carbon nanotube was explored. Due to its unique high aspect ratio structure, the elution order (related to elution mode) can be altered upon the change of length and boundling. Chen et al.  reported using FFFF to separate both single walled carbon nanotube (SWCNT) with a length range of 0.3-4.5 Âµm and multi-walled carbon nanotube (MWCNT) with a length distribution of 3-30 Âµm. Both SWCNT and MWCNT elute out as a broad peak. Fraction at different portion of peak gives rise to different length range of nanotubes. For SWCNT, SEM examination of fractions collected from different portion of peak show short tubes elute out faster than long tubes. This suggests the size range is within the range of flow normal mode FFF. The nanotubes elute out earlier than those 50 nm and 100 nm Nanosphere PS size standards. The length distribution of fractionated SWCNT measured by TEM/or SEM, however, is much greater than the equivalent spherical diameter that calculated from flow normal mode FFF theory. They speculate hollow structure of nanotube and resulting low density may induce them diffuse faster than spheres into high velocity region of parabolic flow profile, resulting in earlier elution of nanotubes. Interestingly, SEM data show the fractionation of MWCNT follows a reverse elution order compared to SWCNT, with short tubes coming out later than long ones, suggesting the MWCNT elute from the channel in steric rather than normal mode. A simulation developed by Phelan Jr. et al.  show nanotubes with an aspect ratio below 1000 (based on tube diameter of 1 nm) should be expected to elute by a normal mode mechanism. Moon et al.  show the use of different surfactants as carrier liquids significantly changes the retention profile of carbon nanotubes.
Gold nanoparticles have been extensively employed in biomedical application such as drug delivery, diagnosis and photothermal therapeutics. Size, size distribution, surface chemistry, aggregation, stability characterization using FFF is very important.
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Song et al. examined 5, 10, 20 nm gold nanoparticles using FFFF and found both partition membrane and mobile phase carrier liquid significantly influence the retention behavior of gold nanoparticles. They compared regenerated cellulose membrane with polysulfone membrane in the presence of nonionic surfactants and concluded the wetting and swelling of membrane contribute to the perturbation of retention. Following their work, Cho et al. recently systematically examined effects of ion strength of liquid carrier, surfactants in mobile phase, accumulation wall membrane chemistry on the retention of gold nanoparticles. They demonstrated high resolving separation capability of FFFF by using a mixture of 10, 20, 30 40 and 60 nm gold nanoparticles. In addition to negative charged gold nanoparticles, the fractionation of neutral charged, PEGylated gold nanoparticles was also discussed. Sermsri et al. studied the size dependence of gold nanoparticle on the concentration of Î±-tocoherol and incubation time. Zattoni et al. investigated the polydispersity of polymer coated gold nanoparticle using FFFF and reported the centrifugation during the surface modification step caused aggregation of gold nanoparticles.
Due to its intrinsic high density, gold nanoparticle was also evaluated by using SdFFF to determine the size, size distribution, and aggregation. The earlier work by Anger et al. demonstrated SdFFF is able to accurately determine the size of gold nanoparticle as small as 20 nm. Contado et al. examined how ratio of citrate and gold affects the size of resulting gold nanoparticles. The size of nanoparticle ranging from 20 nm to 70 nm was determined by SdFFF and confirmed by TEM.
Nano titamium oxide (TiO2) is a type of nanoparticle extensively used in cosmetic products, especially sunscreen. The determination of polydispersity and aggregation of nano TiO2 in wet matrix is of importance because this factors will affect the penetration of . The fractionation and size determination of TiO2 nanoparticle have been reported by Contado et al. [21, 22] and Cardot et al.. in Contado's work, the slurries of nano TiO2 extracted from commercial sunscreen products was analyzed using both FFFF and SdFFF, in conjuction with square wave voltammetry (SWV) and ICP-AES. The size of nano TiO2 was determined based on an average value (4.05 g/mL) of two common crystallographic phases, rutile and anatase, whose densities are 3.84 and 4.26 g/mL, respectively.
FFFF was also applied to the characterization of nano-meter scale imaging agents, such as iron oxide nanoparticles and quantum dots. Lohrke et al.  described the use of FFFF in size characterization of iron oxide nanoparticles. Eight different iron oxide nanoparticles were analyzed and FFFF results strongly suggest the aggregates (multi-agglomerated iron oxide core) observed with a TEM are indeed present in solution, not caused by sample preparation. Polymer coating on the quantum dot surface is critical for improving the water solubility and preventing from metal leaching which consequently cause toxic effects. Zattoni et al.  used FFFF to evaluate the polymer stability by varying the cross-flow rate and found that high higher shear force (under high flow rate) possibly induced the release of the polymer coating.
The advantages of FFFF techniques which include the minimum sample loss, gentle shearing force, sample versatility, field strength programmability were well recognized. It should be noted the stability and inertness of the semi-permeable membrane is a still important factor affecting the application of FFF technique. Some samples tend to interact with partition membrane and therefore cause a secondary effect on retention ratio and instrument reproducibility. Effort toward to minimize such interaction has been made by optimizing partition membrane, mobile phase carrier liquid. A bare frit was also used as the accumulation wall and a 5 Âµm NIST PS microsphere particle was tested to compare the result obtained with membrane .
Figure 1. FFF separation mechanism. (Reprinted from Encyclopedia of analytical chemistry, John Wiley & Son, Chichester 2000, Page 7582-7608)
Figure 2. Schematics of different operation modes of FFF (reprinted from 
Figure 3. FFFF+MALS fractogram of mixture of polystyrene latex bead standards (20, 50 and 80 nm).
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