Reprinted with permission from Pierce Biotechnology, Inc.
Dialysis is a separation technique that gained popularity in life science laboratories during the 1950s. Research papers of that era described dialysis as a new, cutting-edge tool that scientists could use to unravel complex mixtures of biomacromolecules. Many of the dialysis theories established at that time are the corner stones for contemporary products featured in this brochure.
There are, however, two major differences between the dialysis tools of yesterday and today preparation time and
the amount of sample loss due to leaks.
Early laboratory dialysis methods involved dedicating a significant amount of time to membrane preparation, Pierce dialysis products are essentially ready to use and resist sample leakage. New developments in dialysis techniques were stagnant during the last few decades, while ultrafiltration systems flourished due to advances in non-cellulose membranes and to the accessibilit y of bench-top centrifuges. Ultrafiltration via centrifugation was the established convention until Pierce introduced the Slide-A-Lyzer® Dialysis Cassette in 1994.
Dialysis is the separation of small and large molecules in a solution by selective diffusion through a semipermeable membrane. Typically a sample containing a protein or nucleic acid will contain unwanted small molecular weight (M.W.) compounds such as a buffer salt (Tris, PBS, etc.), a reducing agent [Dithiothreitol (DTT), b- Mercaptoethanol (BME), etc.] or a preservative (sodium azide, thimerosol, etc.). The sample is placed on one side of the dialysis membrane. The dialysate, which is 200 to 300 times the volume of the sample, is placed on the other side of the dialysis membrane. This creates and maintains aconcentration differential across the membrane. Once the liquid-to-liquid interface (sample on one side of the membrane and dialysate on the other) is initiated, all molecules will then try to diffuse in either direction a cross the membrane in order to reach equilibrium. Dialysis (diffusion) will stop when equilibrium is achieved. Generally the rate of dialysis slows as equilibrium approaches, requiring that the dialysate in the beaker be changed after several hours to re-create the concentration differential that drives the dialysis process . The membrane is the key to dialysis. The semipermeable membrane contains pores of a known size range that are large enough to let small M.W. compounds pass through, but that are small enough that larger M.W. compounds (e.g., proteins and nucleic acids) cannot get through. The ideal membrane is very thin, has numerous pores of uniform diameter, and is made so proteins and nucleic acids do not bind to it. Unfortunately, the ideal membrane does not exist. What scientists have been using for decades is an extruded regenerated cellulose membrane that has many of the characteristics of an ideal membrane.
However, most scientists often assume too much chromatographic resolution associated with the membranes molecular weight cutoff (MWCO). Pierce determines the MWCO of its dialysis membrane by using the rotating batch dialysis cell (see diagram1 on the previous page). In the rotating cell, the membrane to be tested is held in place between two circular cavities of equal size. One side of the cell is partially filled with a solution containing a molecule of known M.W. The other side is filled with an equal volume of buffer or saline. The solutions are mixed and kept in contact with the membrane by rotating the cell at a constant speed. The M.W. standard concentration in each half of the cell is measured after a fixed period of time and the percent retention is calculated. This type of system provides a more accurate MWCO determination for dialysis membrane than that which is obtained through ultrafiltration methods that measure hydraulic permeability or volumetric flux vs. pressure using saline or buffer alone.
Reference
- Klein, E. et al. DHEW Publication No. 77-1294, p.17.
Other important variables are sample and dialysate volume. The ideal scenario is to have a very small sample volume and a very large dialysate volume, which would maximize the concentration differential. The sample volume is important because subsequent applications have certain minimum volume requirements. However, after the minimum volume requirements are met, it is not advantageous to dialyze 5 ml of sample when only 0.5 ml is needed, wasting the remaining 4.5 ml. Depending on the surface area of a given sample, a smaller volume sample will dialyze much faster than a larger volume sample. Not only is expending additional time wasteful, it can result in sample loss because the longer a sample is in contact with solid-phase surfaces, the more likely it is that proteins or nucleic acids will nonspecifically bind or denature.
