In middle age, field flow fractionation (FFF) is having an identity crisis, although no one seems to notice. Yet, the adventurous early adopters seem to have very positive experiences within a few weeks or months from a cold start. With today’s instruments, the FFF is just not that difficult. And the need is now. Just look at biotechnology and nanotechnology. These must be the top two interest areas in chemistry. There is a crying need for new analytics.
The 4th Annual Eclipse FFF-MALS Focus Meeting was held October 20, the day after the 21st Annual International Light Scattering Colloquium, at The Four Seasons Resort, The Biltmore, in Santa Barbara, CA. Both are open user meetings organized by Wyatt Technology Corp. (Santa Barbara, CA). The meeting gave 19 scientists an opportunity to spend an extra day in Santa Barbara to talk shop. In contrast to prior meetings, the unseasonably cold, wet weather made it much easier to attend the lectures, which were uniformly excellent.
Although FFF encompasses several modes, Wyatt focuses on its Eclipse™ Asymmetric Flow Field Flow Fractionation (A4F) augmented by a stable of detectors. For example, MALS (multiangle light scattering) detection is ideally suited to provide independent and very accurate measurement of molar masses of the samples measured. The Optilab® T-rEX™ refractive index (RI) detector from Wyatt has exceptionally low noise and a dynamic range about 50 times greater than models from other vendors. For a more complete description of the T-rEX, please see “Highlights from the 22nd International Ion Chromatography Symposium (IICS 2010),” at www.americanlaboratory.com/webexclusive/IICS2010.
Chromatography benefits from several competing simulation programs designed to assist the chromatographer with developing and optimizing robust methods quickly. Dr. Dierk Roessner of Wyatt Technology Europe GmbH (Dernbach, Germany) described Isis simulation and optimization software. Isis, named for the Egyptian goddess of simplicity, is intended to facilitate optimization of the control settings and channel construction for A4F. For example, increasing the cross-flow will generally increase retention time of the analytes. Retention can also be improved by increasing the channel thickness. The software provides some error trapping, especially when a setpoint would probably be out of range. The printout from the Eclipse using ChemStation (Agilent, Santa Clara, CA) provides a summary of the plates, resolution, and selectivity for each separation. This is useful in method control and diagnostics.
A4F of polymers
Branching of polymers affects polymer performance, but it is often difficult to measure. A lecture and poster by Dr. Stepan Podzimek of SYNPO (Pardubice, Czech Republic) compared the results obtained from steric exclusion chromatography (SEC) and A4F both with MALS and RI detection for polymers. When the results from SEC-MALS were plotted using the conventional conformation plots of root mean square (RMS) radius vs molar mass over the range of 104–109 Da, the results showed a fishhook shape. The A4F plots were nearly linear over the same range. Dr. Podzimek attributed the abnormal behavior of SEC to the ability of the branches of the molecules to penetrate and perhaps even mechanically anchor into the pores of the SEC stationary phase. As a result, some of the large branched polymers elute more slowly than normal, leading to underestimation of the molecular weight. With A4F, there is no stationary phase, the abnormal elution is eliminated, and the conformation plots are devoid of artifacts. Hence, A4F is useful for measuring conformation and branching.
Dr. Podzimek’s lecture compared A4F with SEC in more detail. The sample in A4F is concentrated during the focusing step, in contrast to dilution only in SEC. The elution order is inverted between the two: With SEC, the largest molecules emerge first, followed by the smaller, in order. For A4F, the elution order depends on the size of the analyte. Over the range of 100 kDa to about 500 nm, which is called the diffusion region, the elution order is small to large. However, at about 1–20 μm, the particles are so large that they extend into the fast current, even when touching the sidewall. This is called the steric region, with an elution order of large particles followed by smaller. The transition or ambiguous zone is for analytes with a particle size near a micrometer or so. One can adjust the channel thickness to change the boundary between the normal diffusion and steric regions or modes. This is one of the attractive features of Isis software.
Several authors, including Dr. Podzimek, found that SEC of molecules larger than about a million daltons is unreliable. In addition to the mechanical delay mentioned above, they also reported that some large molecules may not even elute. Worse, many suspect that the mechanical interaction with the stationary phase is strong enough to break the polymer. This has been confirmed by collection and reinjection.
Large proteins
In addition to the problems with SEC above, Dr. Janice Davis of Althea Technologies (Camarillo, CA) expanded the list of assay problems for large analytes to include dynamic light scattering and analytical ultracentrifugation (AUC). The latter is too slow, and the former needs fractionation prior to the measurement since it responds preferentially to the largest molecules in the sample cell. Because Althea is a contract laboratory, Dr. Davis sees a variety of problems and samples. Some are relatively simple, if one has the right instruments. A case in point: A fractogram with MALS detection of a large multiunit protein showed peaks that corresponded to the expected mass, but some of the peaks presented nonflat responses with the MALS detector, indicating that the peaks were not monodisperse.
Another more extensive study involved a formulation study of an Fc fusion protein. Fusion protein is a protein made by connecting the Fc region of an antibody with another protein segment. The Fc region may reduce adverse host response, and the next region may contain a therapeutic agent. These are often called chimeric proteins since they are designed to combine the best of two species. Large aggregates were of particular concern since they were probably too large for SEC. The first problem encountered with FFF was a sample that overloaded the channel, even with only 100 μg. In an effort to improve detection, the cross-flow was increased from 1 to 3 mL/min. This improved detectability, as expected. Next, aged samples were examined. These showed large peaks eluting after the active pharmaceutical ingredient (API) peak. The magnitude of these peaks was used as a measure of instability to rank-order the formulations, with tall peaks being the worst. Several formulations were much better than the others and passed on to the next stage. Dr. Davis concluded, “Although it remains challenging to use quantitatively, A4F can add value in real-world studies of proteins.”