MWCO filters, also known as centrifugal filters, are commonly used lab ultrafiltration devices for the concentration, purification and buffer exchange of polynucleotides and protein samples. To select the appropriate ultrafiltration MWCO filters, we need to consider many factors, like the starting volume and the molecular weight(MW) of the sample.
Molecular Weight (MW)
In microfiltration, the pore size of the membrane is usually measured in microns(range from 0.1 to 10μm), indicating that particles larger than this specification will be retained by the membrane.
For ultrafiltration membranes(range from 0.01 to 0.1μm ), they are categorized based on NMWL (Nominal Molecular Weight Limit) or MWCO (Molecular Weight Cut Off), which are in the unit of Daltons(Da) or KiloDalton (kDa). 1 kilodalton equals 1000 daltons.
kDa vs Microns
KiloDaltons(kDa) is a unit of measurement for molecular weight. An increase in the kDa of a molecule indicates a corresponding increase in the number of atoms within the molecule. On the other hand, a micrometer (micron or µm) is a measure of molecular length.
Although molecular weight and molecular length are related, there isn't a specific, fixed constant of proportionality between weight and length. This relationship varies due to multiple factors, including the protein's structure, compactness, and conformation in a solution. This means, an increase of atoms, which leads to an increase in kDa value, may still keep the same molecular diameter.
As a general guideline, 100 kDa typically corresponds to a particle radius of 0.01 µm.
For a more precise assessment of a protein's size, techniques like dynamic light scattering (DLS) or size-exclusion chromatography (SEC) can be utilized as they directly measure the size distribution.
MWCO in Ultrafiltration
Molecular Weight Cut-Off (MWCO) is a term used in membrane filtration to describe the pore size or rejection capacity of an ultrafiltration membrane. An ultrafiltration membrane with defined MWCO indicates that 90% of the molecules above this value will be retained by the membrane. Ultrafiltration membrane manufacturers commonly produce MWCO's of 3kDa, 10kDa, 30kDa, 50kDa, 100kDa etc.
However, various factors affect retention capabilities in ultrafiltration, we generally recommend choosing an ultrafiltration membrane with a molecular weight cut-off (MWCO) that is 2-3 times smaller than the molecular weight of the target macromolecule that wants to be concentrated. This choice ensures better results and the efficiency of the ultrafiltration process.
6 kDa < MW < 20 kDa
20 kDa < MW < 60 kDa
60 kDa < MW < 100 kDa
Diagram: MWCO and Target MW
What to learn how to calculate a DNA, RNA or Protein’s molecular weight(MW), Read this article: How to select the correct MWCO for DNA, RNA and protein purification and concentration.
Impact of Small MWCO Filters
Selecting an MWCO membrane that is too small may prolong the concentration process and reduce the flow rate. Additionally, a smaller MWCO filter might lead to protein precipitation due to the high protein concentration. Both excessively rapid or excessive concentrations can contribute to protein precipitation.
Impact of Large MWCO Filters
Conversely, opting for an MWCO that is too large will result in the membrane's surface intercepting more macromolecules and particles in the sample, leading to increased non-specific binding. To maximize yield, it is recommended to use a relatively smaller filter unit and membrane area. Cobetter Briscale® centrifugal filters are made of low protein binding Regenerated Cellulose membranes, which featured a relatively high protein recovery rate.
Figure 1. Protein Recovery Rate of Cobetter Centrifugal Filters
Impacts Affect Interception in Ultrafiltration
As mentioned above, various factors affect membrane retention capabilities and product recovery in ultrafiltration, including molecular conformations, charged properties, sample concentration and composition, operating conditions, and equipment configuration, etc. Here are some factors to take into consideration:
Samples with concentrations below 1mg/mL will pass through more easily compared to those exceeding 10mg/mL. Furthermore, the presence of other molecules can increase sample concentrations as well.
Globular proteins have higher rejection than flexible polymers with extended chains, such as polystyrene and dextran. This implies that linear molecules are more easily to pass through membrane pores compared to globular molecules.
Therefore, for approximately globular molecules, it is best to choose a centrifugal filter with MWCO equal to or slightly smaller than the molecular weight of the target molecule. This will shorten the centrifugation time, ensure the recovery rate simultaneously, and significantly reduce the risk of macromolecular damage.
High transmembrane pressure caused by G-force during the centrifugal process will increase molecule passage. On the contrary, reducing the g-force can enhance the membrane’s molecule retention capabilities.
To prevent swelling or degradation that could impair membrane performance, it's essential for membranes to be chemically compatible with the feed solution.
pH and ionic conditions
pH change and ionic condition change may cause molecule conformational changes or aggregation.
Lower temperatures can reduce molecule permeation through a membrane when compared to high-temperature conditions (4ºC versus 24ºC).
By carefully considering the MWCO of the ultrafiltration membrane, you can achieve optimal concentration, reduce protein precipitation, and enhance overall efficiency in your filtration process. When working with samples lacking a reference, it is recommended to initially test a centrifugal filter with a Molecular Weight Cut-Off (MWCO) that is 1/2 the molecular weight of your target molecule.
As so many factors affect the final result, it is crucial to conduct pilot experiments to validate membrane performance for a specific application. Cobetter Briscale® centrifugal filter units offer reliable performance and high protein recovery, making them an excellent choice for your ultrafiltration needs.