The Dialysis Process

The dialysis process of a solute occurs at the membrane surface, in context to the concentration differences of two solutions on either side of the membrane. This process is affected by the variables of temperature, viscosity and mixing rate of a solution.

The movement of a solute across a semipermeable membrane is the result of random molecular motion. As the solute molecules in a solution move, they will collide from time to time with the membrane until they diffuse. (See diagram 1.)

The permeation of a given solute (Y), from a solution on the left (L) of the membrane to the right (R), and back again, will depend upon the frequency of collisions between Y molecules on either side of the membrane.

For example, if the concentration of solute-Y, in the solution L is 100 mM, and solution R is 10 mM, the probability of Y-solute colliding with the Y-solute in the solution L, will be much higher than the chance of the solute-Y colliding with the R side of the membrane. Therefore, the net rate of transfer of a given solute, (at a certain temperature, viscosity and mixing rate) will increase with greater concentration differences between the two solutions.

Permeation Rate

The rate of transport across a semipermeable membrane depends mainly on the shape, charge, and size of the solute. Speed and size are only two out of numerous interacting factors. The flux, or permeation across a semipermeable membrane of solutes in solution, is inversely related to the molecular weight.

Small molecules collide more often with the membrane, thus, their rate of molecular migration through the membrane will be high. Large molecules, moving at low velocities collide infrequently with the membrane. Therefore, their rate of migration through the semi-permeable membrane will be low (even those that fit through the membrane pores).
The size of a solute correlates highly with the molecular weight. As the molecular size approaches and exceeds the size of the membrane pores (MWCO), passage of solutes will compley or partially be prevented.

Influence on Permeation Rate

• Parameter
• Concentration Gradient
• Molecular Size
• Temperature
• Wall Thickness
• Membrane Surface Area

• Increased
• Large Concentration Differential
• Small Spherical Molecules
• High Temperature
• Thin (<20μm)
• Large

• Decreased
• Small Concentration Differential
• Large Fibrous Molecules
• Low Temperature
• Thick (>20μm)
• Small

Dialysis Membrane Materials

Synthetic and natural membranes are commonly used for filtration applications. Membrane materials most often used include regenerated cellulose, cellulose acetate, polysulfone, polycarbonate, polyethylene, polyolefin, polypropylene, and polyvinylidene fluoride.
The regenerated cellulose in Cellu·Sep® is derived from cotton: Cotton linters are dissolved in a solution and spread into flat sheets or extruded into tubes. The material is treated with glycerin to prevent the pores from collapsing and air dried at a certain temperature and pressure to form a rigid membrane. Cellu·Sep regenerated cellulose membrane has a symmetric pore structure which allows small molecules to migrate in either direction, making it ideal for experimental purposes.

The regenerated cellulose in Cellu·Sep® is derived from cotton: Cotton linters are dissolved in a solution and spread into flat sheets or extruded into tubes. The material is treated with glycerin to prevent the pores from collapsing and air dried at a certain temperature and pressure to form a rigid membrane. Cellu·Sep regenerated cellulose membrane has a symmetric pore structure which allows small molecules to migrate in either direction, making it ideal for experimental purposes.

T-Series Instructions

For most dialysis applications, Cellu·Sep membrane
can be used directly from the roll after a brief rinse in distilled
water.

How Long to Dialyze . . .

Low-molecular weight salts and buffers (e.g.,Tris·Cl and KPO4) equilibrate within 3 hours with stirring. Equilibration times for viscous samples will be longer.
Change the dialysis buffer as necessary. Usually two dialysis buffer changes are sufficient. When CsCl is removed from equilibrium density gradient-banded DNA, two equilibrations against a 1000-fold volume excess of buffer will decrease CsCl concentration 106-fold, to a still-significant 5 µM, and it may be necessary to change buffer a third time.

Protein Adsorption

Cellu·Sep membranes exhibit a very low non-specific protein adsorption when compared with competitive membranes. Cytochrome C solutions exhibit a large adsorptive loss with competitive membranes following a five fold concentration of 150µg/ml solution.

Membrane Area: 14.5 cm2 (43 mm disk), Starting Concentration: 150µg/ml (7.5mg Cytochrome C)

In addition to very low non-specific adsorption the Cellu·Sep membranes have excellent chemical resistance against organic solvents. 

Sensitive Assay Membrane Pre-treatment

If glycerol, sulfur compounds, or small amounts of heavy metals will interfere with subsequent steps, the membrane should be prepared as described below. For convenience, pre-treated, highly clean Cellu·Sep H1 High Grade wet membranes, designed for sensitive applications, may also be used.

1. Wearing gloves, cut the dialysis membrane into the desired length and soak in distilled water for 15 minutes.
2. Heat the pre-cut membrane for 30 minutes, while stirring, to 80 °C in a large volume of 10 mM sodium bicarbonate.
3. Transfer the membranes into a 10 mM Na2EDTA solution and soak for 30 minutes.
4. Replace solution with distilled water and stir for 30 minutes at 80 °C.
5. Allow membrane to cool down, and store in a refrigerator in a 0.05% sodium azide solution, or a 0.10% sodium benzoate solution. Alternatively, a 20-50% ethanol solution may be used. Tubing must always remain submersed.
6. Before use, wash tubing inside and out with distilled water and condition in dialysis buffer (if necessary, tubing may be sterilized).
7. Secure clamp on one end of membrane. Buffer or water should be placed inside the bag to ensure integrity of the seal. Check integrity of the tubing and clamps.
8. Pour out test solution and load sample (For concentrated salt samples, leave space in the tubing to allow for net flow of water into the sample and to prevent tubing from bursting.)
9. Immerse dialysis tubing in beaker or flask containing a large volume (usually 100 to 1000-fold that of the sample) of the desired buffer and dialyze for several hours at the desired temperature with gentle stirring. 
   NOTE: Low-molecular weight salts and buffers (e.g.,Tris·Cl and KPO>4) equilibrate within 3 hours with stirring. Equilibration times for viscous samples will be longer.
10. Change the dialysis buffer as necessary. Usually two dialysis buffer changes are sufficient. When CsCl is removed from equilibrium density gradient-banded DNA, two equilibrations against a 1000-fold volume excess of buffer will decrease CsCl concentration 106-fold, to a still-significant 5 µM, and it may be necessary to change buffer a third time.
11. Remove dialysis tubing from buffer, remove clamp from one end, and remove sample with a pipet.

Membrane Sterilization

The common method of membrane sterilization is exposure to ethylene oxide gas. Alternative sterilization methods are gamma irradiation and steam autoclaving. Several published abstracts illustrate a decrease in permeability after a sterilization process, depending upon the applied method. Below are some of the techniques described.
Suggested preparation of membranes before sterilization is to soak the membrane for 30 minutes in distilled water. After washing, the membrane might be stored in a 0.85% sodium chloride solution containing 0.1% formaldehyde.

Chemical Sterilization with EtO

Place the prepared membrane in an open polyethylene bag in a vacuum oven. Evacuate and fill the oven with a gas mixture of 20% EtO/ 80% CO2 by a total pressure of 1 atm. Treat the membrane for 5 hours at 40 °C. Evacuate the sterilizing gas and admit 50% of relative humidity of air. A slight reduction, approximay 10% permeability characteristic, has been reported with the use of this EtO method.

Gamma-irradiation

Seal the membrane in a polyethylene bag and expose to a gamma ray source for a total dose of 2.5 Megarads. During exposure the temperature should not increase to more than 10 °C. The permeability characteristic after the treatment is approximay 75%.

Autoclaving

The length of the autoclaving cycle should be kept as short as possible. Membranes may be safely autoclaved at 121 °C at 100 kPa (1 bar) for 10 minutes if submersed in distilled water. They should not be permitted to dry out afterwards. Dry heating over a period of 48 hours at 80 °C drops the permeability characteristic to about 50%.

Micro-organism Effects

Membranes, when wet, are most susceptible to microbes and fungi. Such microbial growths will impair the dialysis properties of the material and cause decreased yield and infection of the sample. Therefore, tubing should not be left without satisfactory protection against bacteria and fungi.
Sodium azide and sodium benzoate are commonly used preservatives for serological material. A concentration of 0.05-0.10% sodium azide is recommended for this purpose. However, a considerably lower concentration, around 0.02%, seems to give satisfactory protection, although streptococci growth may occur at this concentration.

Precaution

Sodium Azide (NaN3) is a standard preservative for laboratory reagents. Although NaN3 is considered highly toxic its LD50 in rats is 45 mg/kg orally.
MFPI recommends the following Sodium Benzoate (C7H5NaO2) solution in addition to Sodium Azide (NaN3) for suppressing microbiological growth in wet stored dialysis membranes. Sodium Benzoate is considerably less toxic than Sodium Azide (LD50 in rats of C7H5NaO2 is 4.07 g/kg orally).
Sodium Benzoate is commonly used as a preservative in pharmaceuticals and in food products (not more than 1 in 1000 parts being permitted). Although its preservative effect is best exhibited in slightly acidic media; in neutral media it provides satisfactory protection of the membrane.

Short instruction for preparation of a 0.5 Molar 100 ml Sodium Benzoate Solution:

Reagent: C7H5NaO2, Molecular Weight: 144.04
Dissolve 7.2 grams of C7H5NaO2 in 100 ml of water. The solution may be brought to a boil to speed the dissipation of the C7H5NaO2.

Packing and Storage

All Cellu·Sep dialysis membranes are packaged in unique dispenser boxes to facilitate ease in handling and measuring. The membranes are enclosed in polyethylene bags, containing desiccant pouches to prevent excess moisture and reduce the risk of contamination. The membranes contain glycerol, added as a humidifier and plasticizer to maintain product integrity.
To prevent drying and subsequent brittleness, it is vital for the tubing to be stored properly. Moisture loss can cause loss of membrane flexibility and result in pinholes during handling. In order to avoid this possibility, it is recommended that unused tubing be stored in the original polyethylene bag or any other air tight moisture-proof container in a cool place (refrigerator), in a minimum of 35% relative humidity.

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