Field flow fractionation (FFF) suffered through a long, 40-year infancy and is only now entering puberty. Interest in field flow fractionation is rapidly increasing due to the need for analytical and preparative separations in nanotechnology; very large polymers; and large-molecule therapeutics, including aggregates. The critical size range is 10 nm to 50 μm. Once again, a few years of applications pull are much more effective than decades of technology push.
The 15th International Symposium on Field- and Flow-Based Separations (FFF 2011) was held May 23–25, 2011, and attracted 100 scientists to the South San Francisco Convention Center. Highlights from the meeting follow.
Modes of FFF
Field flow fractionation works with any applied force field. The two most popular are asymmetric flow (AF4) and sedimentation (sdFFF). The former uses a cross-flow through a semiporous membrane, and the latter is usually performed in a circular spinning annulus resembling a centrifuge, which creates a strong centrifugal force field in the annulus. Other forms of FFF include electrical and magnetic fields, which interact with ionic or magnetic materials, respectively. For example, a poster by T.O. Tasci of the University of Utah (Salt Lake City) described a microfluidic magnetic FFF in a 5-cm-long channel with an internal diameter of 30 μm and two electromagnets. Out-of-phase square wave pulses at 1 Hz were applied to the capillary. This produced differential migration of 30- and 50-nm-diam magnetite, with elution times of 7 and 4 min, respectively.
Another poster, from Prof. Michel Martin of ESPCI ParisTech (Paris, France), proposed using standing acoustic waves to focus and separate particles. In another poster, Prof. Phillip Ligrani (St. Louis University, MO) proposed the use of buoyancy-driven separations in which analytes are separated on the basis of relative density in a modified split-flow thin (SPLITT) cell.
FFF and HPLC: The complementary, orthogonal pair
The opening plenary lecture by Prof. Frantisek Svec of the Lawrence Berkeley National Laboratory (Berkeley, CA) showed promising chromatographic separations using nanomaterials in monolith columns—stationary phases incorporating nano-gold, nano-hydroxyapatite, plus nano-fullerenes and carbon nanotubes. The monoliths have throughpores in the low-micron range, so there is potential for handling rather large particles, but chromatography also requires interaction between the stationary and mobile phase. Thus, the interesting surface chemistry usually occurs in the meso- or smaller pores that are not accessible to very large molecules and particulates. This lecture nicely set the stage for FFF, which is gaining a reputation as the technique of choice when analytes are too large for chromatography. In many cases, one needs both; hence they are complementary.
The next lecture illustrated this concept. Tungsten trioxide (WO3) is a band gap semiconductor whose photochemical performance depends on its crystal structure. Prof. Catia Contado (University of Ferrara, Italy) studied the aging of colloidal suspensions of ~25 μm WO3 with SdFFF and AF4. Over a 5-hr period, the particles agglomerate to 120 nm (spherical equivalent), but their morphology is a cluster of square particles. The particle size distribution correlated to the photoelectrochemical properties.
Inorganics and nanomaterials
Several lectures reported using FFF to elucidate the inorganic chemistry of small particles. Prof. Frank von der Kammer (University of Vienna, Austria) studied the complex environmental chemistry of iron and titanium oxides with traces of lead and arsenic. Multiple species are involved. The authors conclude that FFF is an ideal tool to elucidate element–nanoparticle interactions in the 1–100 nm range. Prof. Wenwan Zhong (University of California at Riverside) described using FFF-ICP-MS for the study of proteins located around the corona of nanoparticles. Indeed, based on many posters, AF4 combined with ICPMS appears to be a very popular combination for nanotechnology.
Cancer therapy today is all about risk/benefit ratio for medicines. Dr. Anil Patri of the Nanotechnology Characterization Laboratory (NCL) (Frederick, MD) discussed the challenges in clinical translation of nanomedicine with drug conjugates for cancer therapeutics. In many first-generation chemotherapeutics, less than 0.1% of the injected dose (drug) reaches the target cells. The rest damages the remainder of the host.
Efficacy and safety can be improved by targeting strategies or extending clearance time. While many biodegradable nanomaterials are eliminated from the body if they are smaller than 8–10 nm, some are assimilated (such as iron oxide), even if they reach the liver. However, metallic nanomaterials larger than 10 nm may also accumulate in the liver. Long-term liver toxicities are currently not known for many nanomaterials. While liver retention may not be a concern for patients with advanced cancer, the ideal drug carrier should not cause any additional harm, which requires some manner of clearance or sequestration.
Dr. Patri selected size and surface coverage of the construct as key attributes for drug conjugates. Pegylation is commonly used to increase the biocompatibility, escape the immune system recognition, and decrease the clearance rate. Appropriate polyethylene glycol (PEG) should be chosen. It comes in a range of sizes such as 2000 Da, 20,000 Da, and much larger. Density of coverage is also an important variable.
The Nanotechnology Characterization Laboratory is part of the National Cancer Institute (NCI), a national resource, and provides preclinical assessment of nanomaterials intended as cancer therapeutics. It is a collaboration between the National Institute of Standards and Technology (NIST), the Food and Drug Administration (FDA), and the NCI. The NCL (ncl.cancer.gov) offers critical infrastructure and characterization services to nanomaterial providers.
For example, a poster presented by Prof. Alexandre Moquin (University of Montreal, Canada) prepared a composite particle containing quantum dots and doxorubicin, which is cytotoxic. The quantum dots are for tracking. The outer layer is poly[2-N,N(diethylamino)ethylmethacrylate]–PEG. When the particle is near a tumor, the pH drops due to the high enzymatic activity. This causes the construct to swell, which squeezes out the cytotoxin doxorubicin in the immediate vicinity of the tumor cell, hopefully killing it.
The role of ions, particularly metals between simple ion exchange and river sediments, was often hand-waving conjecture. I recall prior FFF meetings with reports of intermediate species involved in ion transport in surface water. This year, Frank von der Kammer of the University of Vienna reported on studies of nanoparticles that function as ultrafine (dp from 1 to 100 nm) ion exchangers. This report was a work in process. He concludes that elucidating the chemistry of metal ions in surface water will require AF4 with low-molecular-weight cutoff membranes. Steric exclusion chromatography (SEC) may help in the region below 5 nm.
A late afternoon session focused on silver nanoparticles. Silver nanoparticles (nAgs) exist naturally in some waters, but their use as antibiotics adds anthropologic sources, including sewage sludge. Prof. Ehsanul Hoque of Trent University (Peterborough, Ontario, Canada) pointed out a 2009 report that listed 259 consumer products containing nAgs. Prof. Bruce Gale of the University of Utah found that plant microbes try to avoid the toxicity of nAgs with extracellular polymeric substances (EPS). In the presence of EPS, the concentration of nAg must be increased sevenfold to produce the same microbial control.
The concern is the impact these materials may have on health. Prof. Jason Unrine (University of Kentucky, Lexington) is using both sdFFF and AF4 to study silver nanoparticles in soil pore water. He added small amounts of sodium dodecyl sulfate (SDS) to improve recovery. He found no evidence that SDS changed the size of soil or nAg particles. Prof. Hoque uses AF4 with ICP-MS detection to evaluate the fate of nAg in wastewater treatment plants and a sediment system.