Dialysis

Dialysis is the term used to describe the diffusion of solutes through semi-permeable membranes when the membrane  forms  the  boundary between solutions of different concentrations.  The membrane acts as an inert  sieve with a certain  average pore  size.  The pores result from the random distribution of the fibres making up the dialysis membrane.

Figure 1.  The random distribution of (cellulose) fibres in a dialysis membrane.

A “pore” corresponds to a space bounded by fibres. Clearly the pores are not all of the  same size: there  is a normal distribution of pore  sizes. Molecules with a molecular radius larger than  the  largest pore  size of the membrane will be completely  retained  while those  with  smaller radii will pass through more or less easily depending on their size.  Fig. 1  shows a 2-dimensional representation but it must be realized that  pores are 3- dimensional.

Figure 2. Dialysis across a semi-permeable membrane.

With reference  to  Fig.  2,  consider a small  solute  “a”,  initially  in compartment “A” which is separated from compartment “B” by a semi-permeable membrane.  As the  initial  concentration of “a”  in  A  is greater than its concentration in B, a will  diffuse from A-----B .

The rate of diffusion will be affected by the following factors:-

•  The concentration differential across  the  membrane.  Stirring of both solutions, if possible, and regular changing of solution B will ensure that [a]A >> [a]B and thus the  rate  of diffusion  will be kept  at  a maximum.

•  Surface area.  The larger the surface area of the  membrane,  the  faster the overall rate of diffusion.  Therefore the membrane area should always be kept at a maximum.

•  Solution volume.  If the  solute  molecules  have  to  diffuse  a  long distance before reaching the membrane, then  the rate of dialysis will be relatively  slow.  Stirring can  speed  up  the  rate  of  transfer  to  the membrane, but the distance should also be kept to a minimum,  i.e.  the surface area: volume ratio should be large.

Dialysis is typically  used to  desalt protein  solutions,  or to  effect  a buffer exchange,  i.e. to get the protein from one buffer solution into another (Note that “desalting” and “buffer exchange” are really the same process, in the former the second buffer  is simply distilled water).

Figure 3.  Dialysis using a visking dialysis bag.

Dialysis can be done in various ways, but in the  laboratory  it is most commonly done using “Visking” tubing. This is a cellulosic material re-constituted into tubular form, dried, and supplied in rolls. A length can be cut from the  roll, hydrated by  immersion  in water for  several  minutes, and clamped or knotted at one end to form a sealed “dialysis bag”. The protein is introduced into this bag and the open end is sealed by clamping or knotting.  The  dialysis bag is immersed in a large volume  of distilled water or buffer for  several hours at  4C to effect exchange of the permeable ions and molecules (Fig. 3), the dialysis solution being changed at intervals (every few hours).

During dialysis, water enters  the  dialysis  bag  due to  the osmotic pressure of the protein  solution.  For this  reason  a dialysis bag must not be filled, but a potential  space must be  left  to  accommodate  the increasing volume of the protein solution  .  Note that if the  dialysis  bag  is  sealed  with  knots,  the  knot  should  be  tightened  by pulling only on the outside, not on the bag side of the knot,  to  avoid stretching the bag and thus distorting the pores.

1. The Donnan membrane effect

The Donnan membrane effect1 describes the  phenomenon  whereby a charged macromolecule,  constrained  by a  semi-permeable membrane, causes an asymmetrical  distribution  of permeable  ions  on  either  side of the membrane.  The  net  effect  is to  cause an  apparent  movement  of ions, having the same charge as the protein, away from the protein, i.e. if the  protein  is positively  charged, there  will be a lower concentration  of small cations  in the  compartment  containing  the protein  than  in  the compartment on the other side of the membrane, and  vice versa.  In buffers, the  Donnan  effect  is not  very  significant,  but when a protein  is dialysed against distilled water the Donnan effect can cause significant pH differences on either side of the  membrane. This may or may not be significant, depending on the circumstances.

Similarly, ion-exchange resins repel ions of like charge and attract ions of opposite charge.  In buffers  of low ionic  strength,  this  may  cause the pH to be significantly different in the immediate vicinity  of the  resin substituent groups, compared to that  in the  bulk of the  solution,  i.e. cation exchangers, which are negative, will attract cations, including H+ ions, and this will cause a decrease in pH in the immediate  vicinity  of the resin substituents.  With  anion  exchangers,  which are  positive,  hydroxyl ions are attracted and the pH around the  substituents is consequently higher than  in the bulk  solution.

Figure 4. The Donnan membrane effect.

2 . Counter-current dialysis

A very efficient form of dialysis, often used in automatic  analyzers,  is counter-current dialysis (CCD).  In this,  a  stream  of the  solution  to  be dialyzed is arranged to  flow through  a thin,  convoluted,  channel  on  one side of a dialysis membrane. and the dialysing solution  is arranged to  flow in the  opposite  direction  through  a corresponding  thin  channel  on  the other side of the dialysis membrane.  In CCD, a maximal concentration difference is thus maintained between the solution being dialyzed and the dialyzing solution. Since thin channels are used, the diffusion distance is small and so there is little need to stir the solutions.  A stirring effect can be induced, by flowing the  solutions  at  a high  speed  so that  laminar  flow breaks down into  turbulent flow, but the period of dialysis per pass is reduced and the  benefits,  if any,  have  to  be assessed for each case  by empirically establishing the optimal flow rate.

Figure 5.  Counter current dialysis.

3. Concentration by dialysis (concentrative dialysis)

As mentioned above, a “complication” of dialysis is osmosis, which is the movement of water through a semi-permeable membrane from a solution of low osmotic pressure to a solution of high osmotic pressure. Normally the flow is into the protein  solution,  so that  the  protein solution becomes  diluted during dialysis against distilled water or  a buffer solution: for this reason a dialysis bag is never filled when a protein solution is dialyzed.  However,  the  flow can  be reversed  and the  protein solution concentrated, by dialysing the protein  against a solution with a higher osmotic pressure.

The dialysis bag may be simply surrounded by granular sucrose.  The water flowing out of the  bag will dissolve the  sucrose,  generating  a concentrated sucrose  solution with a high osmotic pressure, and this will cause further egress of water from the bag.  Alternatively, the dialysis bag may be suspended in a solution of polyethylene  glycol (PEG), a hydrophilic polymer.

It will be appreciated that, because  water is flowing out of the  dialysis bag in such a case, the  bag can  be filled completely  with protein  solution before concentrative dialysis.  Concentrative  dialysis  is  a  specific  method in the sense  that  only  macromolecules  are  concentrated  - all buffer salts etc. are not concentrated  - but it is non-specific with respect to proteins.

An effect similar to that of concentrative  dialysis can be achieved by adding a dry,  reversibly hydratable  gel (i.e.  one  that  can be dried and reconstituted to have the same structure) such as Sephadex.  The Sephadex xerogel  will absorb water and, provided it  is  larger than  the exclusion limit of the gel, the  protein  will be concentrated  in the  fluid between the swollen gel particles.

4. Perevaporation

A method  of concentration  using dialysis bags but which is not  used much today, is Perevaporation.  In  this  method  a dialysis bag containing the protein  solution to be concentrated  is suspended in a stream  of air. Water evaporates from the outside of the  bag, keeping the  bag and its remaining contents cool.  As  the  water evaporates,  all  of the  non-volatile contents of the dialysis bag are concentrated.

An application of perevaporation which  is frequently used today is in the  drying of polyacrylamide  gels after  electrophoresis.  Dried gels are mechanically strong and are more easily stored than hydrated gels.  To dry the gel, a cellophane membrane  is placed on  either  side of it and the sandwich is suspended in a stream of warm, dry,  air until  it is completely dry.

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 .