Ultrafiltration

Ultrafiltration is a technique related to dialysis, and can also be used to desalt protein  solutions,  effect  buffer exchange,  or  concentrate  protein solutions. It is more expensive than dialysis, however, as special equipment and membranes are required. In this technique, pressure is applied to the solution to cause a bulk flow of water and dissolved low molecular weight solutes,  through  the membrane, while high molecular weight solutes are retained.

Figure 1.  An ultrafiltration cell.

If a conventional  dialysis  membrane  were  used  for  ultrafiltration,  it would soon become  blocked with proteins  trapped  within the  membrane. To overcome this problem, special membranes are used.  These have a unimolecular sieving layer (a layer one  molecule  thick),  supported  by a much thicker support layer having a larger pore size. Such a membrane is called an anisotropic membrane, since it is not the same in all parts.  The sieving side can  be distinguished from  the  support side because the sieving side is shiny while the support side is dull.

Figure 2.  An anisotropic ultrafiltration membrane.

Whether or not a particular molecule will pass through an ultrafiltration membrane is determined at the  unimolecular sieving layer. Proteins which are unable to pass through are rejected on  the  surface where they  can  easily be removed  and the  filter  is therefore  resistant to blocking.

The pressure exerted on the solution causes  a flow of solvent  through the membrane but immediately flow commences, a phenomenon known as concentration polarization occurs. This refers to the process whereby a secondary membrane layer of retained protein is formed.

Figure 3. Concentration polarization: the formation of a secondary membrane layer.

The secondary membrane layer (SML) constitutes the major resistance to flow and thus determines  the  flow rate.  At  any  given pressure, an equilibrium is rapidly set up whereby the  transport  of macromolecules into the SML, by bulk flow of solvent,  is counterbalanced by diffusion of macromolecules  out of the SML.  The SML thus attains a stable thickness and the flow rate remains constant.

If the  applied  pressure  is  increased,  the  flow  rate  initially  increases,  but this results in more  macromolecules being transported  into  the SML. The thickness of the SML thus increases,  its resistance to  flow increases, and the  flow rate  drops,  virtually to  the  original  value.  Thus  the  flow rate is essentially independent of the applied pressure. Since the  resistance  is determined  by the  thickness  of the SML, reducing this thickness  will result in  an  increased flow rate.  One  way  of decreasing the  thickness  of the  SML is to  stir  the  solution  and  thus increase the effective  rate of back diffusion.  This  is the  purpose  of the stirring bar illustrated in Fig. 1.

Figure 4. Dynamic nature of the secondary membrane layer (SML), at equilibrium.

An alternative  way is by the  use  of a thin  channel  ultrafiltration module, in which the UF-membrane is sandwiched between two blocks of Perspex , with corresponding thin channels milled into each (Fig. 5). By pumping the  solution at a high flow rate, turbulent flow can be induced in the  channel and this  stirs up the  SML.  The  pressure  applied  to  the membrane can be adjusted by restricting the outlet  pipe on the  high-pressure side of the membrane. Industrial scale UF is usually accomplished by such flow-through UF modules.  The modules may be stacked, with the flow arranged in series or in parallel, and very high total  membrane  surface areas and overall  flow rates can be obtained.  An advantage  for industrial scale applications  is that such UF systems are one of the few protein fractionation  methods that can run continuously,  all other methods requiring batch wise operation.

Figure 5.  A thin-channel flow-through ultrafiltration module.

The secondary membrane determines the flow rate but the primary membrane (the anisotropic membrane) determines the selectivity, i.e. the size of molecules that  will be retained.  Primary membranes can  be purchased with different “exclusion limits”, ranging from 500-100,000 Daltons. Conceptually, the “exclusion limit” is the molecular weight of a globular protein  which will just be retained  by the  membrane  - which is the same as the molecular weight of a protein which will just pass through the membrane.  In  practice,  however,  there  is  not  a  clear-cut distinction between the  size of molecule which will be retained  and that  which will pass through the membrane, since the pore sizes in any membrane  have  a normal (Gaussian)distribution.

1.  Desalting or buffer exchange by ultrafiltration

Ultrafiltration may be used to change the  buffer in which a protein  is suspended, either for another buffer or for distilled water.  In either case the approach used is the same.  The solution is reduced to a small volume, rediluted in the new buffer (or  distilled water),  reduced to  a small volume again etc., the process being repeated several times, until the protein is in the new buffer only.  The  process is analogous to the washing of the retentate on a paper filter and the same equation applies, i.e.:-

Where

Consequently, to achieve  buffer exchange  (or desalting) in the  minimum time, the factors “u” and “v” should be kept to a minimum, since the time taken depends upon the total  volume of washing solution used. (This is a useful equation to remember when rinsing ones laundry).

2.  Size fractionation by ultrafiltration

Proteins may be fractionated into size groups by ultrafiltration,  by passing the solution, successively, through membranes  of decreasing pore size. The largest proteins will be retained by the membrane with the largest pore size, etc.

References

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .

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مواضيع ذات صلة

Protein (Western) Blotting

Detection, Estimation and Recovery of Proteins in Gels

Cellulose Acetate Electrophoresis

Other Methods of Protein Purification

Chromatographic Methods for Protein Purification

Producing Recombinant Protein

Engineering Proteins for Purification

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production