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Ultrafiltration, Microfiltration (Molecular Biology)
Ultrafiltration and microfiltration are pressure-driven processes that use a semipermeable membrane to separate dissolved and suspended materials on the
basis of size; only molecules smaller than the pore size of the membrane may pass through it. The technique of ultrafiltration can deal with molecules with
molecular weights in the range of 500 to 1,000,00 da, or approximately 10 to 100 A (1 to 10 nm) in size. In contrast, microfiltration uses membranes in which
the pore sizes range from 0.01 to 10 |im (Fig. 1). Ultrafiltration is suitable for concentrating or washing macromolecules such as proteins, peptides,
polysaccharides, lipids, synthetic polymers, viruses, and colloids, or the concentration of dilute samples of nucleic acids. The range of molecular sizes for
which it is used distinguishes the process from reverse osmosis, in which even salts or other low molecular solutes are retained.
Figure 1. Application range for ultrafiltration, microfiltration, and reverse osmosis.
Ultrafiltration cannot discriminate between macromolecules that have sizes similar to one another, and for this reason the process is unsuited for the separation
of soluble molecules whose molecular weights or effective sizes in solution (Stokes radii) differ by less than a factor of 6. For the separation of such molecules,
alternate techniques such as chromatography, electrophoresis, or adsorption should be considered.
1. Dead-Ended and Cross-Flow Apparatus
The ultrafiltration process may be either cross-flow or dead-ended, where the feed stream is forced through the membrane and collected. An example of
dead-end filtration is the disposable syringe end filters that use membranes bonded to a sealed plastic support plate. This unit has Luer-type connectors that
permit its attachment to a syringe. The fluid to be filtered is placed in the syringe, manually driven through the filter, and collected. A technique particularly
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suited for the micro-purification of proteins, antibodies, and nucleic acids is shown in Figure 2. Samples of up to 500 |il may be placed in the microconcentrator
module, which is placed into a centrifuge and spun. The centrifugal force generated forces the filtrate through the membrane and concentrates the molecules
too large to pass through it. For larger sample volumes, stirred cells may be used, such as those developed by Amicon (Fig. 3). In this technique, a flat sheet of
membrane is placed on an appropriate support structure and spacer that forms part of the cell assembly. The fluid is added and agitated with a magnetic stirrer.
The cell is pressurized, either by air or nitrogen, and the filtrate is forced through the membrane. The use of air pressure avoids the damage of highly labile
products that can occur during pumping.
Figure 2. Centrifugal concentrator (MicroconR). After centrifugation, the module is inverted and recentrifuged for more complete recovery of the
concentrate in the vial.
Figure 3. Stirred cell for ultrafiltration. Pressurized vessel with magnetic stirring bar. The membrane in the form of a disk is situated under the
stirring bar.
The main disadvantage of such techniques is membrane fouling. As the filtrate is forced through the membrane, the retained particles accumulate on the
membrane surface. This layer is the concentration polarization layer; its development will depend on several factors, such as temperature, solubility, and pH.
As the thickness of this layer increases, the efficiency of filtration diminishes. Control of such membrane fouling may be achieved by the use of a thin channel
device, in which the feed is forced through a narrow channel that spirals outward over the surface of the membrane. The flow in this narrow channel generates
high shear rates, leading to improved solute transfer across the membrane.
For the filtration or concentration of solutions at process scale, hollow fiber or flat sheet cross-flow devices are used (Fig. 4). In cross-flow filtration
modules, the feed flow is parallel to the membrane and perpendicular to the filtrate flow. The solvent and solutes smaller than the pore size of the membrane
pass through the membrane to form the ultrafiltrate, or permeate. In such devices, the retentate or concentrated solution flows across the membrane when in the
form of a flat sheet, or along the fibers. The permeate or filtrate passes across the membrane and flows out of the module for collection. Cross-flow systems are
inherently more complex, but they have high rates of mass transfer, due to high shear rate or tangential velocity, as well as turbulence adjacent to the
membrane.
Figure 4. Hollow fiber ultrafiltration modules for process scale applications.
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2. Ultrafiltration Membranes
The membranes that are used in ultrafiltration or related processes may be hydrophobic or hydrophilic, uniform in their cross section or asymmetric, with a
dense thin (0.1 to 1.5 |im) top layer supported by a porous sublayer containing macrovoids. The dense surface layer determines the membranes convective
transport properties, since it contains the pores, with the substructure providing mechanical strength. The membranes are made from either cellulose (cellulose
ester or nitrocellulose) or synthetic materials, such as polysulfone and polyacrylonitrile, which exhibit relatively low protein binding.
The basic characteristics of membranes are represented by (1) the flux, (2) rejection of solutes, and molecular-weight cutoff. The flux J is defined as
where Q is the permeated amount, A the membrane area, and Dt the sampling time. The rejection R is calculated from the difference in the concentration of the
solute in the feed solution CF and the concentration in the permeate CP, such that
The cutoff of a membrane is the maximum molecular-weight molecule that the membrane will allow through. For molecules or nucleotides, this parameter is
generally defined as the maximum molecular weight, or the number of nucleotides in the DNA fragment, for which 90% of the molecules are retained by the
membrane.
3. Solute and Solvent Transport Mechanisms During Filtration Processes
The membranes used in ultrafiltration and related processes may be considered a polymeric matrix in which pores are present. Although the actual morphology
of the membranes is complex, considerable insights into the behavior of the membrane may be made by the application of a model to describe the transport
phenomena. As the mean pore diameters of the membranes suitable for ultrafiltration and microfiltration differ, so do the expressions that are used to determine
the flux. For microfiltration membranes, the equations used are based on capillary flow:
The first of these equations is the DArcy flow equation, whereas the second is the Hagen Poiseuille equation. In these equations, J represents the volume flux,
DP the pressure difference across the two sides of the membrane, K the permeability or the DArcy permeability K h the fluid viscosity, and p the universal
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constant. L is the length of the pore capillary, which can be taken to be equal to the thickness of the membrane, since at this level the membrane may be
considered a series of parallel cylindrical pores, with a radius of r, perpendicular to the membrane surface. The equation may also be written to include a
tortuosity factor T in the denominator, which provides a measure of the actual fluid path length through the membrane, relative to the thickness of the
membrane.
For ultrafiltration membranes, the equations for the transport of solvent and solute may be expressed as
where Jv and Js are the fluxes of the solute and solvent, respectively, Lv and Ls their respective hydraulic permeabilities, CM, Cp, and Cs the solute
concentrations in the membrane, permeate, and solution, respectively, DP the pressure difference across the membrane, DP the osmotic pressure, and s the
Staverman reflection coefficient.
Cross-flow filtration techniques are commonly used in which the mixture requiring separation is recirculated over the membrane surface. In this case, the flux
rate will depend on the applied or transmembrane pressure DP, the mass transfer coefficient k of the device, the solute concentration in the formed gel CG, and
the bulk solute concentration CB such that
In the filtration of macromolecular solutions of low concentration by ultrafiltration, their osmotic pressures are low compared to the applied pressure and can
be neglected. In high concentration solutions, the osmotic pressure effects may be significant, and the volume flux equation (5) requires modification to
account for the osmotic pressure of the macromolecular solution. The osmotic pressure exerted by such solution is generally in the form
where A, A^ and A2 are osmotic virial constants and C is the concentration of the bulk macromolecular solution. The coefficient A describes Vant Hoffs
limiting law for the osmotic pressure, which is applicable at very dilute concentrations, whereas A1 can be expressed in terms of the solute molecular weight.
In order to achieve high rates of mass transfer, it is necessary to have a high tangential velocity or shear rate and/or turbulence in the vicinity of the membrane.
Theoretical expressions are available for the derivation of the mass transfer coefficients for differing membrane module and flow geometry configurations.
Such expressions implicitly assume that the density, viscosity, and solute diffusivity are constant across the boundary layer.
Ultrafiltration and microfiltration are able to prepare reasonably concentrated samples of macromolecules, but they permit only a limited separation of the
retained solutes from the smaller or more permeable components present in the solution needing to be filtered. In order to remove such smaller components
more effectively from the retained species, the feed solution can be washed in a process known as diafiltration.
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