Analysis of Protein Aggregates Hydrodynamic Diameter by Field Flow Fractionation (CAT#: STEM-B-0305-CJ)

Field-flow fractionation, abbreviated FFF, is a separation technique which does not have a stationary phase. In field-flow fractionation the field can be hydraulic (with a cross flow through a semi-permeable membrane as the accumulation wall), gravitational, centrifugal, thermal, electrical, or magnetic. In all cases, the separation mechanism is produced by differences in particle mobility under the forces of the field, in a stationary equilibrium with the forces of diffusion: The field induces a downward drift velocity and concentration towards the accumulation wall, the diffusion works against this concentration gradient. After a certain time (called relaxation time) the two forces equilibrate in a stationary equilibrium. This is best visualized as a particle cloud, with all components in constant motion, but with an exponential decrease of the average concentration going away from the accumulation wall up into the channel. The decrease of air pressure going up from sea level has the same exponential decrease which is described in the Barometric formula. After relaxation has been achieved, elution starts as the channel flow is activated. In the thin channel (typical height 250 to 350 µm) a parabolic laminar-flow-velocity profile exists, which is characterized by a strong increase of the flow velocity with increasing distance from the accumulation wall. This determines the velocity of a particular particle, based on its equilibrium position from the wall of the channel. Particles closer to the accumulation wall will migrate slower compared to others being higher up. The ratio of the velocity of a species of particle to the average velocity of the fluid is called the retention ratio R. In FFF for efficient separation, R needs to be below 0.2, typical values are in the range of 0.02 to 0.1.

Biopharmaceutica

Separation in Field flow fractionation takes place in a channel. It is composed of a top and bottom block which are separated by a spacer. The spacer has a cut-out (rectangular or trapezoidal) which creates the channel volume as the spacer is sealed between the blocks. Alternatively, the channel can be milled into the top block as a cavity. The sample is injected in a dilute solution or suspension into the channel and is separated during migration from inlet to outlet as the carrier solution is pumped through the channel. Downstream of the channel outlet one or several detectors are placed which analyze the eluting fractions.

• Sample: Proteins
• Equipment: Field Flow Fractionation

• The formation of aggregates in your biopharmaceutical product can have a negative effect on safety, efficacy and function. Regulatory authorities expect that orthogonal characterization techniques are used to fully understand the aggregation profile of any molecule.
• Measurable range: 1–1000 nm