Asymmetric-Flow Field Flow Fractionation (AF4) is a one-phase chromatography technique. High-resolution separation is achieved within a very thin flow against which a perpendicular force field is applied. The flow and sample are confined within a channel consisting of two plates that are separated by a spacer foil; the plates are then bolted together. The spacer foil has a typical thickness of 100 to 500 µm.
The upper channel plate is impermeable. The bottom channel plate, on the other hand, is permeable, and made of a porous frit material. An ultra filtration membrane with a typical size barrier of 10kD, covers the bottom plate to prevent the sample from penetrating the channel.
Within the flow channel a parabolic flow profile is created because of the laminar flow of the liquid: the stream moves slower closer to the boundary edges than it does at the center of the channel flow. When the perpendicular force field is applied to the flowing, laminar stream, the analytes are driven towards the boundary layer the so-called "accumulation wall" of the channel.
Diffusion associated with Brownian motion, in turn, creates a counteracting motion. Smaller particles, which have higher diffusion rates, tend to reach an equilibrium position higher up in the channel, where the longitudinal flow is faster. Thus, the velocity gradient flowing inside the channel separates different sizes of particles.
The smaller particles are transported much more rapidly along the channel than the larger particles. This results in the smaller particles eluting before the larger ones; the opposite of a Size Exclusion/Gel Permeation Chromatography (SEC/GPC) separation in which the large molecules elute first.
With AFFF separation there is no column media to interact with the samples, so for very high molar mass polymers, there is no need to worry about shearing forces being applied. The entire separation is gentle, rapid, and non-destructive without a stationary phase that may interact, degrade, or alter the sample.
The separation process requires three steps: During the first two steps, injection and focusing, the main flow is split, enters the channel from both ends and is balanced to meet under the injection port. At this point the flow will move only down and permeate through the membrane. When the sample is injected it is focused in a thin band and concentrated towards the membrane. After complete transfer of the sample volume the injection flow is stopped and one typically allows for another minute of focusing before the flow pattern is switched to the elution mode. Now the flow enters only from the inlet port and exits at the outlet which is connected to the detectors. Sample constituents elute separated according to size and are monitored by the array of detectors.
1. Main flow split generates injection flow
AFFF chromatography needs a separate flow to inject the sample into the channel during focusing. Traditionally a separate pump is used to generate the injection flow. This has several down sides. There is the additional expense of an extra pump that needs to be maintained and is a possible source of other experimental problems. Also the pump requires an extra solvent line that adds dead volume and, therefore, makes changing solvent more time consuming. A pump generally needs user attention. The Eclipse has none of these headaches. The injection flow is produced by splitting the main flow delivered through the Agilent pump. A motorized, software controlled valve in combination with a LiquiFlow measuring device delivers the injection flow through the injector into the channel. Advantages are striking: only one pump and one solvent reservoir and one low-pressure solvent line are needed and only one high pressure flow is generated by the pump. The injection flow is computer controlled and measured with ultra-high precision by the LiquiFlow. Wyatt Technology has perfected its proprietary needle valve modification to be a proven, robust, and maintenance free solvent delivery system. All the user has to do is to click on the Inject Flow button or add an inject flow step in the method.
2. Cross Flow regulated with the LiquiFlow device.
In AFFF the cross flow rate regulation is critical because it determines separation. To control the flow rate and change it during a run, either a syringe pump or a LiquiFlow device can be used. Because the LiquiFlow is virtually maintenance free, robust, and stable even after years of use, Wyatt has selected the LiquiFlow as an integral part of the system. The LiquiFlow regulates and measures the flow rate, so that there is an independent check on the accuracy of the separation method. Creating the cross flow always involves a split of the channel flow. The pressure in the channel needs to be high enough to push the solvent at the desired flow rate through the membrane. In fact, if the pressure is too low, the cross flow rate cannot be produced, and a syringe pump does not help. Syringe pumps create cavitation and the correct flow can never be reached. Moreover, there is no feed-back to the user about this problem. With the LiquiFlow system, however, the software shows the actual cross flow at any time. If necessary, the back-pressure on the channel can be increased. With a LiquiFlow, no pulsations or restrictions are introduced, and the cross flow can always permeate the membrane without creating any turbulence. Cross flow rates from 0.1 to 8 mL/min can be created with unparalleled precision. The efficacy of this solution is shown by the superior quality of an Eclipse separation.
3. Minimizing system peaks
During focusing mode there is no flow through the detectors from the channel. The Eclipse produces a bypass flow through the detectors during focusing mode, which is typically set to the same flow rate that is used during elution. This bypass flow is created by a split inside the chassis. It helps to minimize the system peak and perturbations at the start of elution that are created if flow in the detectors is shut down and again turned on during the run.
4. Integration of Agilent 1100 components
One of the biggest innovations in the Eclipse system relates to using HPLC components of a world leading manufacturer. Active control of the HPLC pump is required because flow rates have to be changed during FFF separations. For high productivity and usability, it is necessary to add auto sampler control and control of UV or RI detectors in the FFF software. The Eclipse software has achieved a high level of integration with these Agilent and other components. All relevant functions and features for AF4 are accessible through the Eclipse software so that Eclipse users can benefit from the high performance and quality of the Agilent HPLC pump, auto sampler, and detectors.
5. Channel pressure monitoring
A pressure sensor mounted inside the chassis monitors the pressure in the channel. As the maximum possible cross flow is a function of the channel pressure, changes of channel pressure at constant flow rates are an indication of problems, e.g. plugging or leaks. A pressure signal can be used as a safety measure to shut-down the pump.
6. Motor driven needle valves
Two motor-driven needle valves are installed inside the Eclipse chassis. They offer a significant benefit for the user. The position of the needle valve is computer controlled and can be reproduced exactly by entering the same number in the software. The setting of the focus valve can be changed manually or during a separation run and restored to the original position at any time.
7. Advanced electronic controller with Ethernet communication
The Eclipse software controls the pumps, valves, and the LiquiFlows. It processes input/output signals that are critical for controlling the separation 7 run. This is exactly what industrial process controllers are designed to do, and the Eclipse uses the best of these on the market. The controller has built in intelligence; it can store system values and performs certain control functions independent of the PC. The controller does not show up in the network environment, although it can communicate with its host PC through the existing LAN in the laboratory. This prevents any safety issues, as only the Eclipse software can connect to the proprietary controller interface.
8. New channel design
The channel has been completely redesigned with a PEEK lower block and replaceable inlays for the upper block. The lower block is machined from one piece of PEEK (a high performance polymer that has superior material properties for this job). PEEK is tough, corrosion free, and can be machined with excellent precision. The internal dead volume has been minimized to allow for very fast purging of the block so that bubbles are swept out quickly. The upper block is an aluminum frame that holds an inlay. This inlay, made from polycarbonate or optional from glass, forms the upper channel wall. It can be easily replaced in case of wear or damage. The frit is made from the best available corrosion-free steel and has an unlimited lifetime.
For more information on the Eclipse Field Flow Fractionation system, click here.