Ultrafiltration and Environmental Sampling

Efforts over the last 50 years have triggered the implementation of many laws, amendments, and regulations for environmental improvements.

Recent publications show that scientists and researchers heavily influence efforts for global climate change improvement. Their discoveries guide environmental awareness initiatives and regulations, affecting mandated local, regional, and country-specific testing and environmental health. Freshwater, seawater, soil, sludge, and other samples are studied and graded to track the Earth’s condition. Levels of pollutants, toxins, nanoparticles, genetic material, and proteins are assessed. These results are important for climate control and improvement, and findings may help to avert negative environmental impact.

Ultrafiltration (UF) methods have been incorporated into environmental research and monitoring workflows, with samples prepared from our Earth’s oceans, rivers, waterways, and soils. Small volume samples (0.5 – 70 mL) typically utilize centrifugal ultrafiltration (cUF) devices such as Amicon® Ultra filter units or centrifugal microfiltration (cMF), while large volume samples (up to 400 mL) are compatible with pressurized ultrafiltration (pUF) or pressurized microporous filtration (pMF) devices, such as Amicon® Stirred Cell assemblies. Filtration techniques are widely used in assessing levels of pollutants and evaluating methods for removal, surveying biodiversity in our oceans, identifying biomolecular indicators in soil toxicity, measuring environmental antibiotic resistance, and other studies.

Filtration Device Selection

When using ultrafiltration for sample concentration, particular attention should be paid in choosing the correct filter membrane. Performance may vary by manufacturer, as well as membrane material.

  • Ultrafiltration membranes are typically made from regenerated cellulose (RC) or polyethersulfone (PES). The material of choice will greatly depend on sample compatibility. Ultracel® regenerated cellulose is generally recommended due to lower non-specific binding. PES is used for processing certain plant samples.
  • The selection of membrane nominal molecular weight cut-off (NMWCO) will depend on the molecular weight (MW) of the macrosolute to be retained, and is typically defined in kilodaltons (kDa). This selection will have a significant impact on performance. As a rule, the NMWCO filter rating should be 2-3 times smaller than the molecular weight of the solute to be retained when using Ultracel® regenerated cellulose (RC), and 3-5 times smaller for Biomax® PES membranes.

Generally, solutes larger than the specified MWCO will remain upstream of the filter (i.e., in the retentate), while water and solutes smaller than the specified MWCO pass through the membrane into the filtrate. This can permit removal or retention of other macromolecules that may affect critical biological and chemical determinations, downstream analysis, and assay performance based on size.

When using centrifugal ultrafiltration (cUF) devices, separation, concentration, or washing functions can be performed. Centrifugal microfiltration (cMF) devices are for pre-filtering and washing only.

  • Amicon® Ultra centrifugal ultrafiltration devices use Ultracel® regenerated cellulose (RC) membranes, are offered with nominal molecular weight limits (NMWL) of 3 kDa, 10 kDa, 30 kDa, 50 kDa, and 100 kDa, and accommodate sample volumes of 0.5 mL, 2 mL, 4 mL or 15 mL.
  • Centricon® Plus 70 centrifugal ultrafiltration devices use Ultracel® regenerated cellulose (RC) membranes, are offered with nominal molecular weight limits (NMWL) of 3 kDa, 10 kDa, 30 kDa, 50 kDa, and 100 kDa, and accommodate sample volumes up to 70 mL.
  • Microcon® centrifugal devices use Ultracel® regenerated cellulose (RC) or Biomax® PES membranes. Regenerated cellulose membranes are offered with nominal molecular weight limits of 10 kDa, 30 kDa, and DNA Fast Flow for genomic DNA. PES membrane nominal molecular weight limits include 5 kDa, 10 kDa, 30 kDa, 50 kDa, 100 kDa, and 300 kDa. Microcon® centrifugal devices accommodate sample volumes ≤ 0.5 mL.
  • Ultrafree® microfiltration centrifugal devices for pre-filtering applications use microporous Durapore® (PVDF) or hydrophilic polytetrafluoroethylene (PTFE) membranes, are offered with pore sizes ranging from 0.1 to 5.0 μm, and accommodate samples volumes of 0.5 mL and 2 mL (for PVDF) or 0.4 mL and 2 mL (for hydrophilic PTFE).

Pressurized filtration (pUF and pMF) methods are utilized when centrifuges are not available, for gentler or controlled processing, or to accommodate samples volumes up to 400 mL (this volume can be further expanded with the addition of an external reservoir). The larger sample volume capacity makes pressurized ultrafiltration with stirred cells ideal for larger environmental samples such as seawater, wastewater, or freshwater. Flexible, easy-to-use filter housing assemblies can accommodate a wide range of ultrafiltration and microporous filtration membranes, allowing processing of samples at varying pressures and temperatures. Stirred cell devices for pressurized and microporous filtration are reusable and autoclavable, with magnetic stirring for minimizing concentration polarization and shear stress-induced denaturation during filtration.

  • Ultrafiltration (UF) membranes enable sample concentration and diafiltration, and are rated by nominal molecular weight limits (NMWL) in kilodaltons (kDa). Amicon® Stirred Cell-compatible membrane filters include Ultracel® RC (regenerated cellulose) and Biomax® PES (polyethersulfone), offered in a broad range of NMWL sizes from 1 kDa to 500 kDa.
  • Microfiltration (MF) membranes enable separation and washing, and are rated in microns (µm). Biomolecules or particles larger than the specified rating will remain upstream in the retentate after filtration. Amicon® Stirred Cells can be loaded with a broad selection of disc filters. Durapore® PVDF disc filters are available in 0.1 µm, 0.22 µm, and 0.45 µm for 200 mL and 400 mL Amicon® Stirred Cell units.

Factors to consider when selecting an ultrafiltration centrifugal method or membrane for pressurized filtration methods:

  1. Biological size: This can be estimated from published sources or by various measurement techniques.
  2. Size of key separation targets in solution: The size of sample components (proteins, antibodies, toxins, and other particles that need to be separated) will affect membrane size selection. Refer also to “Membrane NMWL or MF rating”.
  3. Sample volume: Processing volumes ranging from ≤ 0.5 mL to 70 mL are compatible with centrifugal filtration devices. Pressurized ultrafiltration (pUF) Amicon® Stirred Cells have capacities of 50 mL, 200 mL, and 400 mL or more (if external reservoir is used).
  4. Concentration factor or final volume: Final volumes as low as 20 µL can be achieved with Amicon® Ultra 0.5 mL devices. Final volumes as low as 5 µL can be achieved with Microcon® regenerated cellulose devices. Ultrafiltration concentration factor is calculated with respect to starting and final volume. Microporous filtration separates samples without concentration.
  5. Fluid type: Samples may be aqueous, organic, highly viscous, particle-laden, or highly concentrated. In some cases, initial dilution (i.e. buffer or water) may be required. Consult the chemical compatibility or chemical resistance of the device or membrane, referring to the product instructions.
  6. Buffer exchange, dialysis, diafiltration, and washing: Different applications (changing one buffer for another, or removal of salt or chelators) may require different filtration techniques or processes. Multiple spin-wash steps can be performed with centrifugal ultrafiltration (cUF) or centrifugal microfiltration (cMF). Multiple washes can be performed using pressurized systems (pUF and pMF).
  7. Processing conditions: Conditions for centrifugal ultrafiltration and centrifugal microfiltration such as centrifugal g-force (x g), spin time, processing time, and operating temperature may impact performance with centrifugal devices. Conditions for pUF such as pressure (≤ 75 psi), processing times, and stirring rates may impact performance with pressurized devices. Conditions can be optimized as needed. Follow safety guidelines for centrifuges and when using pressurized systems. Centrifugation at lower temperatures will take longer time to process. Nucleic acid purifications should use reduced g-forces. Follow manufacturer guidelines or published methods.
  8. Membrane NMWL or MF rating: For ultrafiltration, to retain a protein, nanoparticle, or other target for concentration, purification, separation, or enrichment, the molecular weight cut-off of the filter membrane needs to be smaller (~2-3 times smaller for regenerated cellulose or ~ 3-5 times smaller for (PES) than the target size, but large enough to allow smaller components to filter through. For small nucleic acids, PCR clean-up reactions, or genomic DNA use 10 kDa, 30 kDa, or Microcon® DNA Fast Flow devices, respectively. (see Table 1). For MF, select a filter with smaller pore size than the particle or biomolecule.
Table 1. Selection of Ultracel® regenerated cellulose ultrafiltration membrane NMWL based on target retention size.
Table 2.Examples of Ultrafiltration in Environmental Research

Ultrafiltration in Averting the Impact of Climate Change

Publications show ultrafiltration and microporous filtration play a critical role in environmental monitoring and research workflows. Applications include eco-analysis for water, seawater, soils, tissues, and biologicals. The utility of Amicon® cUF, cMF, pUF, and pMF devices has been demonstrated for separation, cleanup, and enrichment for lab-scale preparations. Impurities separated for analysis include ionic salts, chemical pollutants, and genomic, molecular, and physical particulate. The optimal filter choice greatly impacts yields, reproducibility of results, and data quality. Both centrifugal filtration (cUF and cMF) and pressurized filtration (pUF and pMF) processes provide quick, simple, and efficient ways to separate larger materials from smaller constituents and concentrate or purify samples. Physical composition of the sample, as well as size and shape of the target retentate or filtrate, are important attributes that need to be considered in filter selection. The Amicon® portfolio, Microcon® portfolio, and Centricon® Plus 70 portfolio offer ultrafiltration devices, assemblies, and membranes for separation, purification, enrichment, desalting, and buffer exchange. Ultrafree®-MC and Ultrafree®-CL filter devices separate by pore size for pre-filtration applications. These devices provide valuable workflow solutions used in studies for the climate challenges of our Earth.

Filtration Products for Environmental Sampling and Research

Table 3.Centrifugal Ultrafiltration (cUF) Product Numbers
Table 4.Centrifugal Microfiltration (cMF) Product Numbers
Table 5.Pressurized Ultrafiltration (pUF) Product Numbers
For use with ultrafiltration or microfiltration membrane discs.
Table 6.Ultrafiltration and Microfiltration Membrane Discs