1 What is ultrafiltration?
Ultrafiltration (UF) is a rapid and efficient separations process widely used in biological research and production. It takes advantage of the high flow rate and selectivity of UF membranes which have a thin shiny "skin" on the top surface. The UF skin has pores of uniform size (much smaller than the structure below) which are cast to be anywhere from about 10 to 200 angstroms in diameter, depending on the size of the protein or other large molecule to be retained. When a protein solution is pushed against a UF membrane, water and buffer ions and other molecules much smaller than the protein flow rapidly through the skin pores. Larger protein(s) which cannot enter the skin pores are rejected and therefore become more concentrated in the retentate chamber as filtrate volume passes through the membrane.
2 What is polarization?
Large molecules rejected by a UF membrane rapidly build up a layer of denser, more viscous solution just above the skin. Unless this layer is removed by mixing or other means, it quickly slows down the rate of filtration and also prevents molecules just slightly smaller than the skin pores from passing through the membrane skin.
3 How can polarization be minimized?
A simple and efficient way is to spin a UF membrane positioned at a sloping angle in a centrifuge to generate filtration pressure. In this situation, the denser polarization layer continuously slides outward across the membrane skin and collects in the bottom of the retentate chamber just as water slides off of a sloping roof.
4 What is fouling?
Lipids, sticky (hydrophobic) proteins, and nucleic acids all tend to adsorb to the skin of many UF membranes, also reducing flow rate and passage of molecules slightly smaller than the pores. Reduction in the flow rate of clean lab water following concentration of a protein solution, compared to initial water flow rate, is evidence of fouling of the membrane. Ultrafiltered lab water such as MilliQ Biocel does not have colloids which can also foul and slow down many UF membranes.
5 How can fouling be prevented or minimized?
UF membranes made from regenerated cellulose are much less sticky than others made from cellulose acetate, polysulfone, or polyethersulfone (PES). The rejecting UF skin of Apollo devices is made from regenerated cellulose.
6 Can fouling be reversed?
Usually, a brief rinse with dilute caustic (~0.01-0.1N sodium hydroxide) and then water or buffer will remove fouling and at least partially restore water flow rate (use appropriate hand and eye protection).
7 What is "cutoff"?
Molecular Weight Cut Off or Nominal Molecular Weight Limit refers to the smallest molecular weight of a typical spherical (globular) protein which will be 90% rejected by a UF membrane of that rating.
8 What are the most common applications for Apollo spin concentrators?
- Concentrate protein in tissue culture media, antiserum or monoclonal antibody preps
- Concentrate protein peaks following gel permeation chromatography
- Desalt/exchange buffer after eluting protein from ion exchange, hydrophobic interaction (HIC), metal chelate or affinity chromatography columns
- Remove unincorporated reagents used to label proteins
- Remove or exchange metals or cofactors in proteins
- Concentrate nucleic acids without ethanol precipitation
- Biosafe method to concentrate virus without over-concentrating proteins in tissue culture media
9 What kinds of research labs use spin concentrators?
- biotechnology discovery
- cell biology
- clinical diagnostics
- molecular biology
- pharmacology discovery
- protein chemistry
- X-ray crystallography
10 How do I decide which UF membrane to use?
Use the QMWL value closest to, but not greater than, the molecular weight of the protein to be retained and purified. If no other, smaller proteins need to be selectively removed, and protein concentration is greater than the mg/mL level, flow rate may actually be faster using a membrane one QMWL size smaller than the protein molecular weight. This is because proteins just slightly larger than skin pores can tend to settle on pores like marbles on a Chinese Checkerboard. See Membrane Selection Guide for more information.
11 Can I use Apollo to remove larger molecules and expect to quantitatively recover a protein that passes through in the filtrate?
This application, known as fractionation, is difficult. Two variables are important to effectively fractionate proteins. The polarization of the larger molecule will tend to increase the retention of the smaller protein. Controlling filtration rate and protein concentration can minimize this effect. Spinning more slowly and diluting the sample beforehand will improve performance.
Another consideration is that the polyethylene microporous support of the composite Apollo UF membrane can tend to adsorb many proteins passing through it as filtrate. If the desired protein in the filtrate is very dilute and hydrophobic, the membrane support material may adsorb much of it before the surfaces become saturated. In this case, prior to use, an innocuous protein or other material may be used to block the binding surfaces in the polyethylene to improve permeate recovery.