Field-Flow-Fractionation

Size: px

Start display at page:

Download "Field-Flow-Fractionation"

Giles Davidson
5 years ago
Views:

1 Field-Flow-Fractionation Calvin Giddings Field-Flow-Fractionation (FFF) is a one-phase chromatography technique invented by Professor Calvin Giddings in The Flow- FFF separation mechanism works based on hydrodynamic forces taking place ina separation channel. The smaller particles are transported faster along the channel than the larger particles and are eluting before the larger ones. W - Channel thickness l - Layer thickness of equilibrium distribution of a component 1

2 Giddings and his team developed Thermal Field-Flow Fractionation in 1969, Sedimentation Field-Flow Fractionation in 1974, Flow Field-Flow Fractionation in 1976 and Split Flow Thin Cell Fractionation (SPLITT) in In 1987 Giddings/Wahlundpublished the first paper about Asymmetric Flow Field-Flow Fractionation, which revolutionized Flow Field-Flow Fractionation and later became the most popular Field-Flow Fractionation technology used today. Another important step in the evolution of the Field-Flow Fractionation technology was achieved, whenmichel Martin first postulated and finally in 1984 published theworld s First Online Coupling of Field-Flow Fractionation with Light Scattering. The method shares some similarities with chromatography, electrophoresis and ultracentrifugation. Retention behavior can not be predicted from chromatographic theory. The ability to produce a high precision channel and controlled field have allowed well defined theories to be developed. Sample components are not partitioned between a mobile and stationary phase. Separations occurs using differential flows of a single solvent in an unobstructed channel. An external field or gradient is applied perpendicular to the flow to drive components into different laminar flows. 2

3 SCHEMA DISTRUMENTAZIONE 3

4 CELLE PER FFF(F) 4

5 As materials are driven towards the channel wall, they move more slowly - basis for FFT separations. Sample is introduced into a solvent stream which enters a thin channel (cell). The forward force is the solvent flow. A retarding force or gradient is applied, driving the sample toward the channel wall. As the sample leaves the center of the stream, it travels at a different velocity. Channel volume 1-5 ml Samples μl Component concentrations are typically 1% or less 5

6 FFF approaches Four common types of fields: Sedimentation Vo/Vr- M and dp Thermal Vo/Vr-D /DT αm -a Hydraulic (Flow) Vo/Vr-D αm -a Electrical Vo/Vr-D / μ Vo - Column void volume dp- particle diameter Vr- Retention volume D - diffusivity M - Molecular weight DT - Thermal diffusivity μ - electrophoretic mobility α- molecular conformation PARTICLE SIZE RANGE 6

7 SEDIMENTATION FFF The channel is spun resulting in acceleration at a right angle to the flow. Field strengths of 40,000 G or greater have been produced. The method can separate colloids in the 30nm to 1μm range. For larger particles (1-2μM) provides an adequate gradient. Gravitational FFF (GrFFF) Subset of sedimentation FFF. Relies on gravity to provide the gradient. Method can be conducted using a modified HPLC. Suitable for measurement of particle size distribution Flow FFF Two crossed flow streams are superimposed on the same channel. Channel walls are permeable and the pore size determines the lower size limit for separation. The driving force is the viscous force exerted on the particle by the cross stream based on sample diameter 7

8 THERMAL FFF A temperature gradient is applied across the channel (80 o C range -1000oC/cm) Particles are driven towards the cold wall. Separation is not as mass selective as sedimentation but has a greater MW range. Steric FFF This approach relies on a different elution mechanism - size. As particle size increases, their motion is halted by the stericbarrier of the accumulation wall. Smaller particles can get closer to the channel wall resulting in a greater effect. 8

9 9

10 Centrifugal FFF La centrifugalfff usa la forza centrifuga come forza di separazione. Le particelle vengono separate in funzione del loro coefficiente di diffusione, in base sia alladimensioneche alladensità. Questa proprietà unica consente di separare particelle della stessa dimensione ma costituite da materiali diversi. Questa tecnica è ideale per la separazione di nano e micro particelle di qualsiasi natura, nanomaterialidi carbonio, emulsioni. SPLITT La SplittFFF usa la forza di gravità per il frazionamento preparativo di microparticelle. Le particelle sono separate in funzione della loro dimensioneedensità, ma anche della loroforma.a seconda della dimensione del canale utilizzato si possono raccogliere da milligrammi a grammi di materiale. 10

11 Asymmetric Flow-Field-Flow- Fractionation (AF4) With Asymmetric Flow-FFF (AF4) the flow and sample are confined within a flat 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. 11

12 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 analytesare 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 AF4/ FFFseparation 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 Electrical FFF A potential gradient is used as the basis of the separation. Initial work separating proteins with different isoelectric points proved promising. Other attempts have used gradient to enhance/control the steric effect 12

13 This new technology combines the principle of Electrical and Asymmetrical Flow FFF in just one system. Electric and Cross Flow Fields can now be applied in one FFF channel. Traditional Flow FFF separations typically provide particle size or molar mass distributions as final result. With the flow and electrical fields overimposed, any of these particle size and molar mass distributions can be further differentiated and transformed into a particle size or molar mass vs. charge distribution. This allows identifying charge heterogeneities which might be present inside the different size and molar mass fractions. As particle and molecule charge plays a primary role in many applications, such as protein aggregation, polymer flocculation, particle agglomeration and also in pharmaceutical formulations in general, this technique opens completely new ways for a significantly better understanding of these phenomena and will quickly help to establish more efficient product development and QC processes Un applicazione della AF4: studio della corona proteica adsorbita su nanoparticelle Anal. Chem. 2013, 85, A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles subsequent behaviors inside the biological systems. Forproteinsbound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. When the equilibrium mixture of the nanoparticleand the proteins was injected as a short plug into the F4 column, the nonbinding proteins would be washed away due to their fast dissociation rates. The nanoparticle-protein complexes were subject to the nonequilibrium separation condition since no proteins were present in the carrier solution of F4. Thus, the complex started to dissociate once the free proteins in the sample diffused out of the sample zone. Then, the proteins dissociating off the nanoparticlewith relatively fast dissociation rates would be found in the eluent at a much lower or even not-detectible content than those with slower dissociation rates, if the initial complex concentration was the same. 13

14 Hollow-Fiber Flow-Field-Flow- Fractionation (HF5) HF5 uses a completely different channel geometry based on a polymeric or ceramic hollow-fiber with porous walls as a cylindrical channel. 14

Dr. Christoph Johann Wyatt Technology Europe GmbH Copyright Wyatt Technology Europe GmbH All Rights reserved 1

Dr. Christoph Johann Wyatt Technology Europe GmbH 2010 Copyright Wyatt Technology Europe GmbH All Rights reserved 1 Introduction Overview The Nature of Scattered Light: Intensity of scattered light Angular