Sample Preparation by Filtration

Chart comparing types of filtration by particle size

Filtration is a separation technique used to concentrate or purify substances based on their physical or chemical properties. It is a simple and routine method used in many laboratories to remove insoluble particles from solutions and to prepare samples for analysis. Filtration is used to reduce sample complexity, improve clarity of viscous samples, and reduce background signals resulting in increased signal-to-noise ratios in analytical tests.  

Depending on the filtration method applied, particles or molecules are separated based on properties such as size, shape or charge. The liquid that passes through the filter is called the ‘filtrate’ and the collected or retained material is the ‘retentate’ or ‘residue’.

  • Reverse osmosis (ionic separation) separates ions or molecules using a semipermeable membrane or barrier. Applied pressure overcomes osmotic pressure and forces solvent to move from a high solute concentration to a low solute concentration. Reverse osmosis rejects a high percentage of organic matter, other than particulates, and >99% of salts. The typical membrane rating is based on sodium chloride retention (<0.001 µm, <100 Daltons)
  • Ultrafiltration (macromolecule separation) separates particles and dissolved molecules from fluids based on particle size. Ultrafiltration is used for concentration, fractionation, desalting, and buffer exchange. The typical rating is a nominal molecular weight limit (NMWL) between 1-1000 kDa.
  • Microporous filtration (microfiltration) is used for particle retention/exclusion and sterilization because it separates/removes particles and biological entities, such as bacteria and cells, based on particle size. Pore sizes are typically between 0.025-10 µm and are rated as nominal (~98% retention) or absolute (100% retention of the size equal to the pore size rating).
  • Clarification filters are used for pre-filtration and particle analysis because they retain/remove large particles, aggregates, and debris based on size. They may be used as a primary filtration step before microfiltration.  Clarification filters typically have pore size ratings >5 µm. retained.

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Common Filtration Applications

  • General particulate removal
  • Sample preparation for analytical techniques such as HPLC, UHPLC, ion chromatography, gas chromatography, and dissolution testing
  • Sterilization of cell culture additives
  • Concentration of proteins, nucleic acids and polymers
  • Separation of biomolecules within a sample
  • Preparation of buffers
  • Water purification

Filtration is an essential sample preparation step before sensitive chromatographic analysis, such as HPLC and LC-MS. Particulates in samples can interfere with liquid, gas and ion chromatography analyses by clogging columns or column heads, or by generating contaminant peaks (“ghost peaks”) on chromatograms. Proper filtration of samples, solvents and buffers generates higher quality, more consistent analytical results. It also increases instrument uptime and prolongs column life.

Types of Filtration Processes & Procedures

There are many filters with different filtration media composition, each designed for particular applications. Filter selection depends on several factors, including:

  • Size of the particles or molecules to be excluded or included
  • Chemical composition of the sample
  • Compatibility of the filtration media with sample or solution
  • Sample viscosity

Filters can be made from different types of materials, such as paper, cloth, cotton-wool, asbestos, slag- or glass-wool, unglazed earthenware, sand, or other porous material. Membrane filters are usually made from synthetic polymers (e.g. hydrophilized PTFE, PVDF, nylon, PES).

Different forces can be applied to drive the filtration process. Filtration can be driven by simple gravity using a filter and a funnel, manually as in syringe filtration, or by centrifugal force. In vacuum-driven filtration, a vacuum pump is used to rapidly draw the fluid through a filter.

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