It often happens that a preparation of macromolecules contains different products that we want to get rid of. These products, salts, carbohydrates, detergents or other small molecules were present in the initial preparation or were introduced during a purification step.
One simple way to remove these small molecules is dialysis. The small molecules that cross the membrane of the dialysis bag are called diffusible. Those that are too large are said to be non-diffusible.
Dialysis is based on the principles governing diffusion through a permeable or semi-permeable membrane. Two mechanisms come into play in this process. First of all, the diffusible molecules will cross the membrane according to the concentration gradient. There will therefore be a net displacement of the molecules from the most concentrated side to the least concentrated side.
Each chemical species in solution individually undergoes this process. At equilibrium, the concentrations of each diffusible species will be equal on both sides. If the volume of the liquid outside the tube is very large compared to that of the solution to be dialyzed, this equality of concentrations implies that the majority of the diffusible molecules, in terms of quantity (weight), is in fact out of the solution. This elimination of diffusible molecules can be amplified by repeating this process.
The other mechanism involves the electrical charges of the molecules. Indeed, the electrical gradient also plays a role. This electrical gradient results from all the species charged on either side of the membrane. The Nerst equation describes this phenomenon. This last factor is however negligible in dialysis and, in practice, only the concentration gradient is taken into account.
METHODOLOGY AND APPARATUS
The solution to be dialyzed is placed in a tube made of a semi-permeable membrane, often cellulose or a derivative. The two ends of this tube are closed, thus forming a “rod”. We place this sausage in the solution against which we want to do dialysis (counter-dialysis liquid).
The porous membranes of the tubes are made of polymerized material obtained by an industrial process. This process leaves residue that must be removed. Generally heating in a solution of sodium triphosphate and EDTA is sufficient.
Dialysis membranes are characterized by an exclusion limit (“cut-off”) which is used to give an idea of the size of the molecules which will not be able to cross it (of mass greater than the limit) and of those which will be able ( less than the limit). Most of the commercially available membranes generally have a limit of the order of 30 kDa. It should be understood that this value is approximate because the size does not vary linearly with the mass, the geometry of the particle is very important. On the other hand, the pores of the membranes are not all identical to me, some being smaller than others. Molecules of mass close to the exclusion limit will therefore probably diffuse more slowly.
There are two types of assembly for dialysis. The first and most common type is closed dialysis. In Place the sausage containing the solution to be dialyzed in a large container. The diffusible molecules are given time to balance on either side of the membrane. This process can be speeded up by shaking the counter-dialysis fluid (e.g. with a magnetic bar). The smaller the ratio between the volume of the retentate and that of the diffusate, the more the diffusible molecules will be diluted during this process. If an even greater dilution is to be obtained, it is sufficient to increase the volume of the diffusate or to repeat dialysis two or three times in a closed environment.
If we want to obtain an even greater reduction in diffusible molecules, we can resort to the second type of arrangement: continuous dialysis. In this technique the rod is exposed to a direct current of the desired solvent, in practice it is most often water. Since the current is continuous, the small molecules will never be able to reach an equilibrium concentration and will constantly diffuse outside the dialysis bag. We can thus virtually reach a zero concentration of these molecules.
It should be remembered that the dialysis solvent and the conditions must be compatible with the stability of the molecules of interest contained in the dialysate.
More recently, devices have been developed for microdialysis which also make it possible to concentrate the solution to be dialyzed. This device essentially consists of centrifuge tubes fitted with a dyalise membrane. By centrifuging, the small molecules and the large majority of the aqueous solvent are forced through the membrane and the concentrated and free macromolecules can be collected on the surface of the contaminant with a mass less than the exclusion limit (“cut- off “), of the order of 5 to 10 kDa.
The vocabulary of this technique has evolved, particularly under the influence of hemodialysis (renal dialysis) which is a medical application of the same basic principle. The term dialysate, in particular, is confusing, often due to the influence of English; for some authors it means the contents of the sausage, for others, on the contrary, it refers to the counter-dialysis liquid containing the diffusible molecules. It would be better not to use this term anymore.
The solution resulting from dialysis, that is to say the solution remaining in the rod and rid of diffusible molecules, is often called retentate. Indeed it is retained in the bag. The counter-dialysis liquid containing the diffusible molecules can be called diffusate, because it diffuses out of the bag, or perfusate.
The goal of dialysis is to reduce the concentration of small molecules in a mixture to a very low level. The diffusible molecules will cross the membrane without obstacle of the dialysate in the counter-dialysate. At equilibrium, their concentrations will be equal on both sides of the membrane.
For these molecules, it is therefore in fact a “dilution” where we would put a small volume of one solution in a large volume of another. For these small molecules, the calculations are done in the same way as if we calculated a simple dilution! If we do successive dialyses, we obviously have to recalculate each “dilution” one after the other to know the final concentration of the diffusible molecules. If you do continuous dialysis, you have to assume that the “dilution” is infinite. In either case, the concentration of non-diffusible molecules will not change.