6.  Dialysis and  Ultramembrane  Filtration





A. Principle :


Dialysis is a separation process depends on the differential transport of solutes of different sizes across a porous barrier separating two liquids where the driving force is  concentration gradient only. It is usually used to separate solutes too large to diffuse through the barrier from those small enough to diffuse through it.




a.Spectra / Por Dialysis membrane.


b.Closures for Dialysis membrane


c.0.01 M Sodium phosphate buffer, pH 7.2 (Appendix - I)


d.Antibody to be desalted (Human Ig)


e.Magnetic stirrer and magnetic bar.


C. Method:


1.Cut the required length of flat dialysis membrane and place in distilled water.


2.Open the membrane  by  gently  squeezing from  one  end  to  another and  wash the inner  side of membrane by passing the distilled water gently without damaging the membrane.


3.Close one end of the membrane using closure or thread.


4.Pour the antibody solution through another end and close the same.


5.Place the filled bag in a beaker containing 0.01M SPB and magnetic bar and keep the beaker on a magnetic stirrer and adjust the speed (at 4 0C).


6.Change the pre-cooled buffer after every 4-6 hr for three time (1 lit each time)


7.Collect the antibody.




Note :


1.Choice of the pore size of the membrane depends on the substance to be dialysed.


2. For  critical  work  such  as enzyme studies etc. extensive cleaning of the membrane is necessary before use.


3.The membrane should not dry because it may damage the membrane.


4.It is advisable to test the system for leaks using distilled water before adding the protein solution to the tubings.




Ultramembrane  Filtration


A.Principle :


  Ultrafiltration sometimes called reverse osmosis is a process in that the solutes and solvent, up to a certain critical size are forced through the barrier by high pressure on one side of the porous barrier than the other. Since there is always a flow of solvent moving along the solute it can be used for the concentration process.


B.Materials :


a.Ultrafiltration tubing.


b.Closures for the tubing.


c.Conical flask with side arm.


d.Funnel and single-holed rubber cork.


e.Sample to be concentrated.


C.Method :


1.Cut 10-15 cm ultrafiltration tubing and wash it as mentioned in Dialysis.


2.Insert the tip of the funnel at one end of the tubing  and  pass the  end  of funnel  with membrane through the rubber cork.


3.Close the other end of tube with closure or with thread.


4.Pour the sample to be concentrated through the funnel.


5.Apply vacuum pressure through the side arm and close it. Leave the flask at 4 0C.


6.Collect the sample by opening the tied end and dialyse if necessary.