Field river fractionation

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The field-flow fractionation ( English field-flow fractionation ; abbreviated: FFF) is a technique of analytical chemistry . Field-flow fractionation shows parallels to liquid chromatography and gel permeation chromatography . However, the separation does not take place in columns, but usually in river channels.

properties

Typical applications are the analysis of nanoparticles , macromolecules such as synthetic polymers , biopolymers (e.g. polysaccharides ) and proteins . An advantage of all FFF systems is the separating field that can be freely adjusted using the software. Thus, different samples can be measured one after the other without changing the column. There are hardly any interactions or shear forces in the FFF systems; thus the systems are suitable for the most difficult samples; a high-temperature FFF system, for. B. Polyethylene analyzed. Another form of field flow fractionation is the hollow fiber FFF (HF5).

history

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

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

principle

The Field-Flow Fractionation is a separation technique, with different variants. These FFF variants all use the same general separation principle, but they differ in the application of different separation fields or forces. Depending on the separation field used, one speaks of flow field-flow fractionation , sedimentation field-flow fractionation , thermal field-flow fractionation or gravimetric field-flow fractionation . More recently, the hollow fiber field flow fractionation (HF5) has also been further developed. Instead of the flat membrane in the separation chamber, the HF5 uses a hollow fiber with a round cross-section in a plastic cartridge for the separation process. There is also a preparative variant called Split Flow Thin Cell Fractionation (also SPLITT Field-Flow Fractionation ). Overall, the FFF method offers a quick, gentle and high-resolution separation of proteins, polymers, biopolymers and particulate substances in liquid media in the size range from 1 nm to 100 µm and 1 kDa up to the megadalton range. The separation area thus far exceeds that of a single chromatographic column. The separation takes place without a column in an open, flat separation channel with laminar flow and which does not contain any stationary phase. Due to the parabolic flow velocity profile within the duct, the absolute flow velocity increases from the upper or lower side of the duct towards the center of the duct, with the highest flow velocity in the center of the duct.

Depending on the variant of field-flow fractionation used, different separation fields are used, such as B. a second liquid flow ( Flow FFF ), centrifugal forces ( Sedimentation FFF ), temperature gradients (Thermal FFF) or just the gravitation of the earth ( Gravitational FFF ). These separating fields are usually applied at right angles to the laminar channel flow. Under the influence of these separating fields and the opposing diffusion of the particles to be separated, a dynamic balance of forces is established. The driving force behind this diffusion is Brownian molecular motion. Smaller particles move more strongly, larger ones less. For small particles with strong intrinsic diffusion, the equilibrium position is therefore spatially higher in the flow channel than for larger particles with low diffusion. Because of the parabolic flow prevailing in the channel, the small particles are in faster flow lines on average over time and are therefore eluted from the channel before the larger particles. This leads to an elution profile that is the opposite of that used in size exclusion chromatography; That is, the small particles appear first, followed by the large particles. If you couple the FFF separation with chromatography detectors, such as. B. light scattering and absorption photometers, refractive index measurement, fluorescence spectroscopy or mass spectrometers, so-called fractograms are obtained. The specialty of a fractogram in the flow FFF is that the peaks represent an increasing particle size or a larger molar mass with increasing retention time , because the separation in the flow FFF is only based on the effective diffusion coefficient and not on the interaction between one mobile and a stationary phase, as is the case with chromatography.

Building an FFF system

The essential components of an FFF system are shown below using the example of the widely used asymmetrical flow field flow fractionation (AF4). The basis of the separation principle are the orthogonally acting fluid flows. These can be generated in different ways. On the one hand, you can use several pumps. A pump generates the detector flow, i.e. the constant flow through the separation channel. A second pump in this system ensures the cross flow, which is directed perpendicular to the detector flow and exits through a frit located on the channel bottom. Another pump is used to focus the injected sample, in which the particles are arranged in the separation channel according to their diffusion coefficient.

The individual rivers described above can also be generated with just one pump. The flow coming from the pump is split up by high-precision valves and variably distributed between the detector flow and the cross flow. The control of these processes is computer-assisted, whereby the system works quickly, precisely and without disturbing the flow and pressure conditions. In some cases it may be necessary to work without metal or with organic solvents. Both are made possible without any problems through the use of special components.

In order to avoid the formation of disruptive gas bubbles during the separation process, the solvent is passed through an inline degasser, which removes dissolved gases. The sample can be injected either manually or using an auto-sampler. In the latter case, larger numbers of samples can also be processed automatically, which leads to a considerable increase in productivity.

After the separation, the components can be collected in fractions. As a rule, however, they reach the detectors where the characterization takes place.

Separation systems

A basic distinction is made between five systems.

  1. River field flow fractionation (F4)
    1. Symmetrical flow field flow fractionation (SF4), in which the top and bottom of the channel consists of permeable frits through which the cross flow is directed
    2. Asymmetrical flow field flow fractionation (AF4), in which one usually uses a channel that has a solid impermeable wall as the upper limit. As with symmetrical flow field flow fractionation, the lower channel boundary consists of a porous frit with a membrane on top
    3. Hollow- fiber flow field-flow fractionation (HF5 ): hollow fiber made of semi-permeable material replaces the separation channel. High sensitivity at low flow rates and volumes, so it can also be easily combined with mass spectrometry as a detection method.
  2. Sedimentation field flow fractionation (SF3), which uses a rotating ring channel, where the separation field is generated by centrifugal force, also called centrifugal FFF
  3. thermal field flux fractionation (ThFFF), which uses thermal diffusion to create retention

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

Detectors

The detectors used are refractive index (also RI detectors from English refractive index ), ultraviolet (UV) and infrared (IR) detectors, as well as viscometers and light scattering detectors . A general distinction is made between the so-called concentration detectors, the signal of which is proportional to the concentration (RI, UV and IR), and the molecular mass-sensitive detectors (viscosity, light scattering). Static light scattering detectors, also known as SLS or MALS, are suitable for determining the molar mass and radius of gyration . Online coupling with detectors based on the principle of dynamic light scattering is suitable for determining the hydrodynamic radius . B. can determine the particle size of micro- or nanoparticles . For some time now, coupling with mass spectrometry with inductively coupled plasma ( ICP-MS ) has also been used to determine the particle size-dependent distribution of the elemental compositions.

calibration

Conventional calibration using a concentration detector: Polymer standards with low polydispersities are used for calibration. The result is relative molar masses .

When using a concentration detector in conjunction with a viscosity detector: For calibration, polymer standards with low polydispersities are used and a calibration curve Log (molar mass × intrinsic viscosity) is established. Since the product of (molar mass × intrinsic viscosity) is proportional to the hydrodynamic radius, the relative or absolute molar masses can be calculated.

Light scattering detection

By using a light scattering detector, there is no need to set up a calibration curve. The light scattering detector measures the absolute molar masses directly. A concentration detector is also required for evaluation. The Rayleigh 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 mass M w . Here, K is an optical constant and A 2 is the second virial coefficient. With multi-angle light scattering, the intensity of the scattered light is measured simultaneously from several angles and the molar mass is determined using the data using linear regression . This makes it clear that the measuring range is larger and the determined values ​​are more precise, the more measuring points, i.e. angles, are used for the determination. It is important to mention that the molar masses are determined absolutely, i.e. H. without calibration or reference to standards. The most powerful devices therefore work with up to 18 angles. But not only the number of angles used is important. The signal-to-noise ratio of the system is also decisive for the quality of the measurement . This means that very precise measurement results can also be achieved with 3-angle devices when the molar mass is small.

further reading

Web links

Individual evidence

  1. ↑ a b Christoph Johann, Stephan Elsenberg, Ulrich Roesch, Diana C. Rambaldi, Andrea Zattoni, Pierluigi Reschiglian: A novel approach to improve operation and performance in flow field-flow fractionation . In: Journal of Chromatography A . tape 1218 , no. 27 , July 8, 2011, p. 4126-4131 , doi : 10.1016 / j.chroma.2010.12.077 , PMID 21227436 .
  2. ^ J. Calvin Giddings: A New Separation Concept Based on a Coupling of Concentration and Flow Nonuniformities . In: Separation Science . tape 1 , no. 1 , 1966, p. 123-125 , doi : 10.1080 / 01496396608049439 .
  3. ^ GH Thompson, MN Myers, JC Giddings: Thermal field-flow fractionation of polystyrene samples . In: Analytical Chemistry . tape 41 , no. 10 , 1969, p. 1219-1222 , doi : 10.1021 / ac60279a001 .
  4. ^ J. Calvin Giddings, Frank JF Yang, Marcus N. Myers: Sedimentation field-flow fractionation . In: Analytical Chemistry . tape 46 , no. 13 , November 1, 1974, pp. 1917-1924 , doi : 10.1021 / ac60349a046 .
  5. ^ JC Giddings, FJ Yang, MN Myers: Flow-field-flow fractionation: a versatile new separation method . In: Science . tape 193 , no. 4259 , September 24, 1976, p. 1244-1245 , doi : 10.1126 / science.959835 .
  6. ^ J. Calvin Giddings: A System Based on Split-Flow Lateral-Transport Thin (SPLITT) Separation Cells for Rapid and Continuous Particle Fractionation . In: Separation Science and Technology . tape 20 , no. 9-10 , 1985, pp. 749-768 , doi : 10.1080 / 01496398508060702 .
  7. ↑ General Theory about Field-Flow Fractionation
  8. ↑ Absolute Molar Mass Characterization. Retrieved May 6, 2011.
  9. ↑ Christoph Johann, Thomas Jocks: The hollow fiber field flow fractionation (Hf5): Improved performance in the separation and characterization of complex protein mixtures . In: LC / GC AdS. 6, No. 1, 2011, pp. 12-16.