Filtration

are leaders in . The Whatman range of multiwell devices is centred around an extensive range of filter microplates and associated solid bottom collection/storage plates. In order to specify the most effective filter microplate an understanding of the filtration process is required.
Filter media have different properties that affect the performance of the filter in microplate applications. When selecting a filter media, the following properties are considered.

Depth Filtration vs. Surface

A depth filter traps contaminants both within the thickness of the filter and on the surface of the filter. Cellulose and are with no defined pores, unlike a membrane that is a with a defined . Cellulose filter papers and glass fibres consist of a matrix of intertwined fibres in which particulates may be trapped in as well as on the surface of the filter. Since a depth filter does not have defined pores it is characterised not in terms of pore size, but in “particle retention”.

The advantage of a depth filter is that since entrapment and adsorbtion occur within the upper fraction of the media, there is a considerable surface area available for filtration. Also, because of its random matrix of fibres retains a large percentage of particulate. The disadvantages of a depth filter are that in the case of a sudden surge of , as can occur when a vacuum is suddenly applied, the filter media will slough off fibres or particles during the filtration period. Also particulates trapped within the matrix can be forced through the matrix and contaminate the filtrate. In many applications a depth filter is used as a to clean a sample. A good example is the Whatman glass fibre filter paper GF/B with a thickness of 675 um and mean pore size of 1.3µm.

A screen membrane traps particulates larger than its rated pore size on its surface. Particulates smaller than the specified pore size may either pass through the membrane or may be captured within the membrane. The advantages of a screen membrane are that rigid particulates can form a porous cake on the surface of the screen membrane and effectively improve the throughput of the screen. Also there is little risk of the filtrate being contaminated. The major disadvantage of a screen membrane is that the filtration process is slow compared with a depth filter

Membrane filters are used for critical applications such as sterilising and final filtration. An example is the PVDF membrane with 0.2µm pores size.

A “combination filter” combines different membrane pore sizes or combines depth media and membrane filters to create a serial filtration units. These multi-layer filters can offer an economical alternative to using individual prefilters and final filters.

A double filter membrane can have unique characteristics not achieved by either of the constituent single membranes. The filter membrane must be capable of retaining liquids during the application. Hydrophobic filters may be appropriate. Examples of such situations are:

  • Cell capture prior to assay
  • Removal of bacterial bio-load
  • Retention of precious liquids

Chemical Compatibility

Chemical Compatibility is defined as the ability of a filter media to resist select chemicals, to prevent damage to the pore structure and the filter material. This also prevents the shedding of particles or fibres To select the proper filter media and microplate housing, the compatibility of the filter with the fluid must be established. Temperature, concentration, and length of exposure time affect chemical compatibility.

The materials used in the manufacture of filter media are carefully chosen for their resistance to a wide range of chemical solutions. An understanding of the compatibility between the fluid to be filtered and the filter elements is essential.

Hydrophilic vs. Hydrophobic

Hydrophilic filters possess an affinity for water. Hydrophilic filters can be wetted with virtually any liquid and are the preferred filter media for aqueous solutions. Hydrophobic filters repel water. A hydrophobic filter will not wet in water but will wet in low surface tension liquids such as organic solvents. Once a hydrophobic filter has been wetted out by an organic solvent, aqueous solutions will pass through.

Both hydrophobic or hydrophilic GF/C and PVDF filters are available.

Pore Size

The pore size of a filter is the diameter of the particle that is retained by the filter. Pore sizes are measured in micrometers (µm). Pore size ratings refer to the size of the particle or organism retained by the filter. Porosity is the percentage of all of the open spaces (pores) in the membrane. Generally membranes are 50% to 90% open space.

For microplate applications a nominal pore size is quoted. A nominal pore size rating describes the ability of the filter to retain 60 to 98% of the particulates that are equal to or greater than the rated size. Process conditions such as concentration of contaminant have a significant effect on the retention efficiency of filters.

Note: Nominal Pore Size Ratings vary widely among different filter manufacturers.

Some of the more common molecular biology particles filtered and their size are:

  • Microbial cells 10µm to 0.3µm
  • Viruses 0.3µm to 0.02µm
  • Blood cells 8.0µm to 3.5µm
  • Yeast’s 4.0µm to 0.6µm
  • Proteins 0.5µm to 0.0005µm

Extractables

Extractables are contaminants that elute from the filter media or device that may adversely affect the quality of the filtrate. These contaminants may include wetting agents in the filter media, manufacturing debris, sterilisation residuals, adhesives or additives in polymer or housing components, colorants, mold release agents, etc.

Polypropylene contains total additives of 0.5% or less. Leaching out of extractables occur at temperatures over 50°C. The type and amount of extractables vary with the type of liquid being filtered. Extractables can affect filtration in almost every application. In cell culture they can kill cells. In microbiological analysis they can affect the recovery of microorganisms. Glass fibre is chemically inert.

Binding

Binding is the ability of a substances to interact with the filter media. Binding is desirable when an assay calls for the nucleic acid or protein binding on the filter. For example the natural hydrophobic PVDF membrane has high molecular DNA or protein binding. Other high binding filters are cellulose nitrate and nylon. However, binding can also be undesirable if proteins indiscriminately bind to a filter during the filtration of a proteinaceous solutions This can result in the loss of active ingredients during filtration. Hydrophilic PVDF has low protein binding. Other low binding filters are polypropylene and cellulose acetate

In addition to the generic ability to bind, the membrane must have the capability of immobilising the component of choice specifically without interference from others.

The membrane should immobilise the molecule in such a manner that orientation and confirmation result in optimal biomolecular activity.

Thermal Stability

Thermal Stability is the maximum temperature that the filter media and microplate remain stable. Thermal stability is important when considering sterilisation by autoclaving Polystyrene cannot be autoclaved but polypropylene can. There is a relationship between chemical compatibility and thermal stability. Many types of filter media are compatible with a chemical at room temperature, but not at elevated temperatures. Most filter media are stable up to 100°C when exposed to aqueous solutions. If organic solvents are used the max. temperature could be as low as 50°C.

Flow Rate

Flow rate and throughput are two important performance factors that are affected by the following variables.

Differential Pressure

This is the difference between the pressure on each side of the filter. A high pressure on one side forces the filtrate through the filter to the lower pressure on the other side. This occurs during filtration in a vacuum manifold. As the filter begins to clog, differential pressure increases.

Viscosity

This determines a liquid’s resistance to flow. The higher the viscosity of a liquid (at a constant temperature and pressure), the lower the flow rate and the higher the differential pressure required to achieve a given flow rate.

Membrane Porosity

This is the measure of all of the open spaces (pores) in the membrane. Generally, membranes have 50 to 90% open space. Flow rate is directly proportional to the porosity of the membrane. More pores equal higher flow rate.

Filtration Efficiency

This is not just a factor of pore size. Other factors are porosity, pore density, tortuous path, and hydrophobicity. e.g. Hydrophobic PP has a slow flow rates to prevent analyte breakthrough when a vacuum is applied.

Selecting a Filter Microplate

As mentioned earlier a clear understanding of membrane characteristics is essential to identify the right filter for assay and process applications. To summarise, the characteristics to consider when selecting filter multiwell devices are:

  • Retention Capabilities Binding characteristics
  • Maintenance of Flow Rates Pore Size
  • Filtration Efficiency Chemical Compatibility

The filter MUST:

  • Bind component of choice (high specificity)
  • Bind component in a biologically active state
  • Be compatible with detection mode (high sensitivity)
  • Have tensile strength to withstand the rigors of the protocol.

Technical Reference – Filter Types

Filter Papers
Whatman qualitative and quantitative filter papers are, with few exceptions, manufactured from high quality cotton linters which have been treated to achieve a minimum alpha cellulose content of 98%. These cellulose filter papers are used for general filtration and exhibit particle retention levels down to 2.5 µm. There is a wide choice of retention/flow rate combinations to match numerous laboratory applications.

The different groups of filter paper types offer increasing degrees of purity, hardness and chemical resistance. Whatman quantitative filter papers have extremely high purity for analytical and gravimetric work.

Membrane filters allow the efficient retention
of sub-micron pariticulates organisms.

Glass Microfibre (GMF) Filters
The unique properties of borosilicate glass microfibres enable Whatman to manufacture filters with retention levels extended into the sub-micron range. These depth filters combine fast flow rate with high loading capacity and retention of very fine particulates. Due to the high void volume exhibited by glass microfibre filters, the choking life is considerably extended beyond the life of a cellulose filter of similar retention. Whatman glass microfibre filters are manufactured from 100% borosilicate glass and are completely binder free. Binder free glass microfibre filters will withstand temperatures up to 550° C and can therefore be used in gravimetric analysis where ignition is involved.

Glass Microfibre filters are manufactured
by Whatman from 100% borosilicate glass.

Membrane Filters
Unlike cellulose and glass microfibre depth filters, membrane filters are conventionally classified as surface filters because the filter matrix acts as a screen and retains particulates almost entirely on the smooth membrane surface. The retention levels for these filters extend down to 0.1 µm and allow the efficient retention of sub-micron particulates and organisms. Water microbiology and air pollution monitoring are major applications of membranes.

Whatman cellulose filter papers exhibit
particle retention levels down to 2.5µm.

Prefilters
The life of a membrane filter can be extended many times by placing a prefilter upstream of the membrane. The total particulate load challenging the membrane is considerably reduced thus allowing the membrane to operate efficiently.

Multigrade GMF 150 combines two filters
in one fast, effective multi-layered filtration.

Filter Selection Guide

Users should verify compatibility based upon experimentation with a specific filter under actual-use conditions.

Flow Rate is directly related to pore size (1 = Low, 5 = High).

Nitrocellulose

General Comments:
Highly adsorbitive membrane, typically used for DNA/RNA/protein hybridisation, also for ELISA and RIA based assays. Available in a wide range of pore sizes with supported and reinforced variants.

Membrane Type – Nitrocellulose
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
4 High Yes Poor Brittle <125

Polysulphone

General Comments:
Limited range of pore sizes. Available in reinforced formats. Very low protein binding.

Membrane Type – Polysulphone
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
4 Low Yes Poor Brittle <120

Cellulose Acetate

General Comments:
Typically used for bulk filtration, good wet strength. Limited range of pore sizes. General purpose microbiological filter.

Membrane Type – Cellulose Acetate
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
3 Low Yes Moderate Moderate <180

Polycarbonate

General Comments:
Precise pore sizes available in a wide range. True surface capture membrane. Translucent/transparent. Tissue culture grades available. Sensitive to bases and aromatic or halogenated hydrocarbons.

Membrane Type – Poycarbonate
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
1 Negligible Yes Moderate Good <140

Polypropylene

General Comments:
Typically used for prefiltration. Limited range of pore sizes available. Sensitive to gamma sterilisation. Very low extractables, chemically inert.

Membrane Type – Polypropylene
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
3 Negligible No Very Good Good <80

PTFE – Polytetrafluoroethylene

General Comments:
Typically used for gas venting. Limited range of pore sizes available. Naturally very hydrophobic, low extractables, chemically inert. Sensitive to gamma sterilisation.

Membrane Type – PTFE Polytetrafluoroethylene
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
4 Low No Very Good Good <130

Nylon (Activated)

General Comments:
Careful storage required. Material inherently hygroscopic. Activated formats can have very high specific binding characteristics but may require careful handling. Limited range of pore sizes available, typically in reinforced formats.

Membrane Type – Nylon (Activated)
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
3 High Yes Very Good Good <135

PVDF – Polyvinylidene Fluoride

General Comments:
Limited range of pore sizes available. Low protein binding, good chemical resistance.

Membrane Type – PVDF Polyvinylidene Fluoride
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
4 Low Yes Good Good <135

GMF – Glass Microfibre

General Comments: Wide range available. Typically used as absorbtive or adsorbtive wicking media and prefilters. Physically weak but available with chemical binders. Good particulate retention.

Membrane Type – GMF Glass Microfibre
Flow Rate Protein Binding Hydrophilic Solvent Resistance Physical Strength Thermal
Resistance (C)
5 Negligible Yes Good Poor High
Basic Principles of Microfiltration
Microfiltration can be defined as the separation of particles of one size from particles of another size in the range of approximately 0.01 µm through 20 µm. The fluid may be either a liquid or a gas.

Microfiltration media are available in a wide variety of materials and methods of manufacture. They can be rated either “absolute” or “nominal” depending upon the percentage of capture of particles of the same size or larger than the retention rating of the media.

Membrane filters are generally rated as absolute media. They can be manufactured of various polymeric materials, metals and ceramics. Nominal media includes filters made of glass fibers, polymeric fibers, discrete particles (diatomaceous earth), ceramics, etc. However, even absolute media can be considered absolute only within a finite time span because of the possibility of bacterial grow-through.

Microfiltration membranes can be divided into two broad groups based on their pore structure. These are membranes with capillary-type pores, hereafter called screen membranes, and membranes with tortuous-type pores, hereafter called depth membranes.

Figure 1
Screen membrane filters

Figure 1 shows a scanning electron micrograph of the surface of a screen, or capillary pore, membrane. This membrane has nearly perfect round cylindrical pores, more or less normal to the surface of the membrane, with even random pore dispersion over the surface. Screen membranes are absolute and are commercially available in thin films of poly-carbonate and polyester. They are manufactured in a two step nuclear track and etch process. They are preferred in a wide variety of applications including optical and electron microscopy, chemotaxis, exfoliative cytology, particulate analyses, aerosol analyses, gravimetric analyses and blood rheology.

Figure 2

How polycarbonate screen membrane filters are made: In the first step, thin plastic film is exposed to ionizing radiation forming damage tracks. In the second step, the tracks are preferentially etched out into pores by a strong alkaline solution.

Figure 3
Depth membrane filters

Figure 3 is a scanning electron micrograph of the surface of a typical depth, or tortuous pore, membrane. This membrane has a relatively rough surface where there appears to be many openings considerably larger than the rated pore size. Depth membranes are nevertheless absolute, depending upon the random tortuosity of their numerous flow paths to achieve their pore-size rating. Depth membranes are commercially available in pure silver, PVC, PVDF, PTFE, various cellulosic compounds, nylon, polyethersulfone, polypropylene and many other materials.

Most depth membranes are manufactured of various polymeric materials using a casting machine. Membranes cast with cellulosic esters are the most widely used membranes. Referring to figure 4, cellulosic membranes are manufactured by dissolving the cellulose esters in a mixture of organic solvents; adding various chemical agents for improved characteristics; and casting the solution as a film approximately 150 µm thick onto a moving belt. As solvents are evaporated under controlled conditions, the tortuous pore structure is formed. The resulting open area ranges from 75% to 89%. Membranes of this highly-porous structure with its labyrinth of interconnecting isotropic pores are recommended for general precision filtrations, electrophoresis, sterilization of fluids, culturing of microorganisms and for many other uses.

PTFE depth membranes are manufactured by the controlled stretching of a fluorocarbon sheet. Some polypropylene membranes have also been manufactured by this method.

The silver membrane is manufactured of pure metallic silver particles that are molecularly bonded to each other to form a uniform porous monolithic structure. A major application for silver membrane filter is inorganic material analyses.

With the difference between screen and depth membranes, it is clear that the characteristics of the two types of membranes would allow each to have significant advantages and disadvantages. For optimum results, membrane users should consider all characteristics in selecting which (or both) of the two types of membranes should be used.

Particle Retention

Particles are captured directly on the surface of the screen membrane. However, screen membranes retain with certainty only those particles the same size or larger than the pore size of the membrane. Except for inertial impaction and diffusion, most particles smaller than the pore size pass unimpeded through the screen membrane.

The screen membrane should also be selected if the user wants low non-specific binding (maximum yield of particles or proteins in the filtrate). This is important, for example, when viruses are being separated from a growth solution and the maximum yield of viruses is desired. Binding of proteins in screen membranes has been found to be less than 10 percent that of depth membranes.

With depth membranes, most particles are captured within the interstices of the membrane except for relatively large particles. Since depth membranes depend upon the tortuosity of their flow paths for capture, they will trap not only particles of the same size or larger than the rated pore size, but also many particles below that rated pore size.

Should the user want maximum removal of all particles and /or a high binding capacity, the depth membrane should be selected. The depth membrane has a much larger available surface area than the screen membrane; therefore it has a much larger particle loading capacity and many more sites where proteins and viruses can bind.

Flow Characteristics

Screen membranes have no side-to-side flow due to their capillary pores, therefore they are unsuitable for electrophoresis and other applications requiring this characteristic. Depth membranes have excellent side-to-side flow.

Flow rates for the two types of membranes are roughly equivalent. Although the depth membrane has more open area, the screen membrane is thinner, 10 µm vs. 50 to 120 µm.

Other Major Characteristics

Both membranes are non-migrating. Both types of membranes can be autoclaved. Cryogenic temperatures have little or no effect on either membrane.

The screen membrane is completely non-hygroscopic. Many depth membranes absorb moisture from the air and must be dried before use, especially in some critical analytical procedures.

Both screen and depth membranes are generally hydrophilic except for PTFE and polypropylene which are inherently hydrophobic. Both types of membranes can be made partially or completely hydrophobic.

Chemical resistance of the various kinds of membranes depends upon the material from which they are manufactured. PTFE has the best chemical resistance, followed by PVDF, silver, polyester, polycarbonate, and finally the various cellulosic compounds.

Common dyes do not stain or discolor polycarbonate or polyester screen membranes; however this must be considered for membranes manufactured of cellulosic compounds.

Major Characteristics of Screen Membranes

– Pore size and structure are well defined

– Particle size cut-off is sharply defined

– No media migration

– Smooth, flat surface for SEM, TEM and optical analysis.

– Non-hygroscopic

– Thin, retains little liquid

– Low adsorption, low absorption

– Low non-specific binding (3-10 µgrams/cm²)

– Non-staining

– Strong

– Repeatedly autoclavable

Major Characteristics of Depth Membranes

– Large surface area

– High dirt-loading capacity

– Long life

– No media migration

– Good handling characteristics

– Repeatedly autoclavable

– High binding capacity (100-250 µgrams/cm2)

Filters for the Pulp and Paper Industries

Hytrex® Filters Fill Multiple Needs for the Pulp and Paper Industry

The pulp and paper industry has several applications for Hytrex cartridge filters that are often overlooked. First, Hytrex filters can be used for filtration of bleaching chemicals (methanol, chlorate, and sulfuric acid) prior to mixture. These chemicals are mixed to form chlorine dioxide, the primary chemical in the paper bleaching process. Any contaminants can cause spotting or other flaws in the final paper product.

Hytrex filters can also offer effective protection for high-pressure spray nozzles. These nozzles are used at several critical points in the paper making process. One such area is the drying section of the paper machine, where the paper is blotted by a series of felts that are continuously cleaned by high-pressure spray nozzles. If a nozzle plugs, and one portion of the felt is not sufficiently cleaned of paper fibers, paper quality is compromised.

High-pressure water showers are also used to clean wire screens following the head box of the paper machine. In this application, a mixture of 99% water and 1% wood fiber flows onto a moving screen to form a continuous sheet of paper. The purpose of the screen is for dewatering at a specified rate. It is important to keep the wire screen clean to avoid the risk of an off-quality product or paper breakage. Generally, filters with a micron rating of one-sixth the diameter of the nozzle orifice are suggested.

A more critical application for Hytrex filters is filtering the raw materials for paper coatings. Raw materials such as binders, pigments, and water all contribute contaminants to the coating process.

Case Study

A large pulp and paper plant in western Canada has selected Hytrex cartridge filters as an affordable alternative to absolute rated filters for chemical filtering, pump seal protection and felt showers. Excellent performance and reasonable price dictated the change to Hytrex filters.

Periodic tightening of the paper market means that economical filtration alternatives, like Hytrex filters, are the best choice for the cost conscious engineer. For absolute filtration, Selex® depth filters are also an affordable option for high quality paper production.