Field flow fractionation

The field-flow fractionation ( english field -flow fractionation; abbreviated FFF) is a technique of analytical chemistry. The field-flow fractionation is parallel to the liquid and gel permeation chromatography. The separation takes place here but not in columns, but usually in river channels.

Properties

Typical applications include the analysis of nanoparticles, macromolecules, such as synthetic polymers, biopolymers (such as polysaccharides) and proteins. One advantage of all FFF systems is about the software freely adjustable divider panel. Thus, different samples can be measured in succession without changing columns. In the FFF systems hardly interactions or shear forces occur; Thus, the system for the most difficult samples are suitable to a high-temperature FFF system is analyzed, for example, polyethylene. Another form of field-flow fractionation, the hollow fiber FFF ( HF5 ).

History

The technique was in 1966 by John Calvin Giddings (* 1930, † 1996) invented and patented at the University of Utah in Salt Lake City, USA.

Giddings conducted research among others in the field of chromatography. However, he was known for his work in the field of field-flow fractionation. He was the founder of the " Field-Flow Fractionation Research Center" ( FFFresearch Center ) at the University of Utah. Where he developed and described, together with its employees and colleagues in several publications, " Theory of Field - Flow Fractionation " and also most of the previously known variants of Field-Flow Fractionation. Giddings and his team developed there first the Thermal Field - Flow Fractionation (thermal field-flow fractionation) in 1969, followed by sedimentation field- flow fractionation ( sedimentation field-flow fractionation) in 1974, the flow field flow fractionation ( flow - field-flow fractionation) in 1976 and finally the Split flow Thin Cell fractionation ( SPLIT ) 1985. founded in 1986 by Giddings company to commercialize the FFF technique FFFractionation was merged in 2001 with the company Postnova.

Principle

The Field-Flow Fractionation is a separation method with different variants. This FFF variants all use the same general principle of separation, but using different separation fields or forces. Depending on the separation field, the term therefore by Flow Field-Flow Fractionation, Sedimentation Field-Flow Fractionation, Thermal Field - Flow Fractionation Field-Flow Fractionation or Gravimetric. There is also a preparative variant which SplitFlow Thin Cell Fractionation (also SPLITT Field-Flow Fractionation ) is called. Overall, the FFF method provides a fast, gentle and high-resolution separation of particulate substances in liquid media in the size range of 1 nm to 100 microns and 1 kDa to in the megadalton range. The separation column runs in an open, flat, laminar flow separation channel, which contains no stationary phase more. Due to the parabolic flow velocity profile within the channel, the absolute flow rate of the channel top and bottom channel takes her to the canal towards the center point, and in the center of the channel there is the highest flow rate.

Depending on the used variant of field - flow fractionation separation of different fields are used, such as a second liquid stream ( flow FFF), centrifugal forces (sedimentation FFF), temperature gradient ( thermal FFF), or even the earth's gravity ( Gravitational FFF). These release agents are usually applied fields perpendicular to the laminar channel flow. Under the influence of these fields and separating the oppositely directed diffusion of the particles to be separated itself provides a dynamic balance of forces. For smaller particles with greater self-diffusion, this equilibrium is spatially higher in the flow channel than for larger particles with lower diffusion. Due to the prevailing in the channel parabolic flow, the smaller particles are averaged over time in faster flow lines and eluted temporally before the larger particles from the duct. If you couple the FFF separation by chromatography detectors such as mass spectrometers, light scattering and absorption photometers, refractive index measurement or fluorescence spectroscopy, called fractograms are obtained, which are similar to evaluate a chromatogram. The special feature of a fractogram however, is that the peaks with increasing retention time increasing particle size or increasing molar mass represent, since the separation in the Field-Flow expires quantity-based Fractionation and not based on the interaction between a mobile and a stationary phase as is the case with the chromatography.

Building a FFF system

The basic components of a system are FFF one to four pumps, injection system, channel separation, and various detectors. The pump draws in the eluent, and generates a constant flow through the separation channel while a separating field prevails. Often, the eluent is drawn through a so-called inline degasser, which removes dissolved gases. After the pump is the injection system, either manually or by an autosampler. Here, the sample finds its way into the system. In the subsequent separation channel, the sample is separated depending on the separation field according to their properties ( hydrodynamic radius, molecular weight ). The various detectors provide then depending on certain statements. In an analytical separation of the entire river lands (including sample) in a waste container. The river can also be collected in a preparative separation in individual vessels.

The simple systems that use only a pump, less user-friendly, failure-prone and maintenance- intensive than those in which multiple pumps are used. The advanced 3- pump technology all required flows are not generated by the division of a single pump flow. 3 -pump systems use FFF - optimized pump with a very constant and finely controllable flow rates. In addition, only systems without mass flow controllers, ie those that use three pumps, metal real freedom can generate, since the sample at any time come into contact with metal. Modern three -pump systems use a separate focus in the port channel for focusing the sample. This is independent of the outlet port to prevent a reverse flow of current in the channel is in focus. Instead, the focus will flow slowly and steadily lowered and built to the same extent of laminar flow. The pressure conditions in the channel always remain constant. A " slosh " effect and thus turbulence of the sample, which would cause a deterioration in the separation outcome is prevented. Moreover, no valves are required in the flow path to the detectors, which can in turn clog the sample.

Separation systems

A distinction is made between five systems.

The AF4 is currently the most widely used form of field-flow fractionation, while SF4, SF3 and ThFFF is used in more and more applications.

Detectors

Are used as detectors, refractive index ( RI detector also ENGL. Refractive index), ultraviolet ( UV ) and infrared detectors (IR ), and viscometer and light scattering detectors used. A general distinction in the detectors, the so-called concentration detectors whose signal is proportional to the concentration (RI, UV and IR ) of the molecular mass sensitive detectors (viscosity, light scattering). For the determination of molecular weight and radius of gyration of static light scattering detectors, which are also known as SLS or MALS are. For the determination of the hydrodynamic radius, the online coupling with suitable detectors that can determine, for instance, the particle size at micro - or nanoparticles according to the principle of dynamic light scattering. For some time, the coupling with the inductively coupled plasma mass spectrometry (ICP-MS ) is used for determining the particle- size-dependent distribution of the elemental compositions.

Calibration

Conventional calibration using a concentration detector: For calibration available polymer standards with low polydispersities. The result is relative molar masses.

When a concentration detector in combination with a viscosity detector: For calibration polymer standards are used with low polydispersities, and a calibration curve log ( intrinsic viscosity molar mass x ) set. As the product of ( molar mass × intrinsic viscosity) is proportional to the hydrodynamic radius, the relative or absolute molar masses can thus be calculated.

Light Scattering

By using a light scattering detector setting up a calibration curve is not necessary. The light scattering detector directly measures the absolute molar masses. For evaluation a concentration detector is also required. Rayleigh 's equation describes the Rayleigh scattering, or the relationship between the scattered light intensity, which is expressed by the so-called Rayleigh ratio R ( θ ), the polymer concentration, C, and the weight average molecular weight Mw. Where K is an optical constant and A2 the second virial coefficient. In the multi- angle light scattering intensity of the scattered light is measured simultaneously from multiple angles and determined by the data by linear regression, the molecular weight. This makes it clear that the measuring range, the greater and the values obtained are more exact, the more measurement points, ie angle, are used for the determination. It is important to mention that the determination of the molecular weights is absolute, that is, without calibration or reference to standards. Therefore, the most powerful devices operate at up to 18 angles. But not only the number of angles used is important. Critical to the quality of the measurement is also the signal -to-noise ratio of the system. So you can also be achieved with 3- angle devices very accurate results when it comes to small molecular weights.