Diagram 1: The most important FFF methods

Diagram 2: Separation range of different analytical methods compared to FFF

Diagram 3: Analytes characterized with FFF

3 Separation principle

Separation in FFF occurs in a thin flow channel, which is comparable to the separation column used in chromatography. In general this channel is approx. 30 cm long, 4 cm wide and it has a height of 250 mm. The channel flow, of an aqueous or organic solvent, carries the sample through the channel. Because of the low channel height this flow is laminar and therefore called the "laminar flow". Perpendicular to this channel flow a second force is generated (diagram 4).

Diagram 4: Schematic illustration of the general separation principle of FFF

In AF4 a liquid flow called "cross flow" is used for generating the second force field. The exact principle of the separation is shown in diagram 5.

Diagram 5: Separation principle of Asymmetric Flow-FFF

Differently sized particles with varying diffusion coefficients are separated by the velocity gradient inside the channel. After the injection the particles/ polymers are forced in the direction of the lower membrane by the cross flow. The cross flow leaves the channel through this membrane, whereas particles and polymers are rejected by the membrane. Smaller particles will diffuse back into the channel further than larger particles because of their larger diffusion coefficients. As a result smaller particles are located in the area of faster channel flow stream lines inside the channel and in this way will be eluted from the channel before larger particles, that are located in slower stream lines and eluted later.

The upper channel wall is liquid impermeable and made of PMMA or glass. The cross flow is generated by dividing the laminar flow, that is pumped in the channel, into two partial flows. One of the partial flows, correspondingly the laminar flow, leaves the channel with the separated sample at the outlet leading to the detectors. The second partial flow, the cross flow, exits through the lower channel wall, which is made of a frit with an overlaid membrane. Using this asymmetric type of cross flow has the advantage of a less complicated channel construction, i.e. no top frit as compared to Symmetric Flow-FFF. Additional separations are improved relative to symmetric F4. Diagram 6 shows the different separation steps of AF4.

Diagram 6: Principle of separation steps used in Asymmetric Flow-FFF

The separation based on AF4 technology, is divided into 4 single steps. These are injection, relaxation, focusing and elution. The first three steps injection, relaxation and focusing are quite simultaneous and are followed by the elution. In the first step, the channel flow is split and introduced both at the inlet and at the outlet of the channel. The relation between the two partial flows is tuned, so that both flows meet each other in the area of the injection port. There they exit through the membrane at the bottom. During this process a liquid funnel is generated, where the sample is injected and simultaneously focused and relaxed. The formerly circular injection spot is compressed to a band during the focusing. Because of focusing the band broadening of the sample peaks induced by injection can be reduced significantly. But this is only one reason for the better separation results of AF4 in contrast to other Flow FFF systems. Another advantage, for example, is the possibility of a on-channel concentration using the injection step. After a few seconds of focusing and relaxation, the regular elution is started, in which the channel flow is only introduced at the inlet. In this step the particle fractions are carried through the channel through to the outlet on to the detection systems.

4 Instrumental Set-Up

The instrumental Set-Up of a AF4 system is comparable to a HPLC system. But the fractionation of samples takes place in a separation channel instead of a separation column. An illustration of the HRFFF 10.000 series, developed by Postnova Analytics, is shown in diagram 7.

Diagram 7: Illustration of the Postnova AF4 system (HRFFF 10.000 series)

In contrast to the Symmetric Flow FFF only one pump is needed in AF4 to create the channel flow as well as the cross flow. A further pump, the injection pump, is necessary to inject the sample. Additional valves, a precise flow-measuring control unit and electronics for the automatical operation and the control of the different flows during separation are needed. These technical components are all integrated in the FFF module of the HRFFF 10.000 series, which was developed by postnova analytics especially for this purpose, that everything necessary for operating an AF4-System, is combined in one compact and bench space saving module. None of the valves or other sensitive components are left outside the system on laboratory benches, where they are unprotected and hinder working.

Diagram 8: General construction of an Asymmetric Flow-FFF channel 

Usually an asymmetrical separation channel has a length of approx. 30 cm, a width of 6 cm and a height of 5 cm. The material of the lower channel block depends on customers requirements. Available are PMMA or other materials (PEEK, stainless steel etc.). Integrated in the channel block is a precision ceramic frit with a pore size of approx. 5 mm. On top of the frit are an overlaying membrane and a spacer. The upper channel wall consists of a special, very flat glass or PMMA plate, with holes and ports for the inlet and the outlet of the channel flow as well as for the sample 
injection. The spacer serves both to determine the thickness of the channel as well as to seal the channel construction.

5 Applications

As stated above AF4 is suitable for fast and gentle separation and characterization of complex particle and polymer systems. Multiple applications have been realized. For special applications you may want to take a look at our application notes, the "Novasheets". Some examples are added to this method description.

If you didn´t find your special application, we are happy to discuss your specific needs so that we can send you the relevant Novasheets. We also offer further possibilities to investigate the FFF-Technology.

• Plan a visit in our application center in Munich or Salt Lake City. We are able to show you the FFF-Technology in action and demonstrate the fields of application and the handling of the technology.

• We can analyze your samples after consultation with application department and so demonstrate the efficiency of the technology from your real sample systems.

• Participate in a seminar for FFF (1-day-training) in Munich. The training seminars include an extensive introduction to the theory and practice of the FFF from our qualified references, as well as the hotel costs and an impression of our nice Munich.

For further detailed information contact us at the following addresses:

6 Performances of Asymmetric Flow FFF

Asymmetric Flow FFF in general, and especially the new Postnova HRFFF 10.000 series, have a number of advantages that distinguishes it from other common separation systems and FFF technologies. 

ADVANTAGES OF AF4:

• No preparation of the sample necessary - direct injection of unprepared samples.
  

• Large dynamic field of masses and sizes can be analyzed. 
  

• Very gentle conditions of separation without a stationary phase.
  

• No or low shear forces because of the absence of a stationary phase.
  

• Rapid times of analysis ranging from 1 to 20 minutes.
  

• Minimal generation of artifacts unlike the GPC (no size exclusion limit).
  

• Less unwanted interactions/adsorptions of the sample because of small surface: FFF separation channel approx. 30 cm2, GPC column approx. 30.000 cm2.
  

• Automatic sample concentration during injection of the sample. 
  

• Variable and flexible method of separation - wide range of eluent buffers and detectors.
  

• AF4 is ideal for analysis of complex colloid, particles or polymer systems because problems as polarization of concentration, blockage of pores and particle coagulation play a secondary role.