Today, the modern life science laboratory is equipped with more sophisticated instrumentation, increasingly automated to enable higher sample throughput. However, no matter what analytical techniques are being used, accuracy of results is largely dependent on the quality of sample preparation; and the correct choice remains fundamental to successful analyses. Dr Barbara Gledhill reports.
Filtration still remains one of the most essential and widely used analytical and preparatory techniques, The reason for this is simple. Manufacturers such as Whatman pay great attention to evolving their own product ranges in line with the changing demands of emerging new technologies.
As a result they maintain an extensive range of high-performance filtration media, supplied in a comprehensive choice of housings.
However, selecting the correct filter for a particular application need not be daunting, providing that important factors are taken into account such as the size, nature and concentration of the particles to be removed, sample volume, flow rate and whether the application is automated. Chemical compatibility of the filter media with the solvent is also crucial.
Particle retention
Filter media are available offering various chemical compatibilities, and are classified according to whether particle retention occurs primarily on the surface (in the manner of a sieve) or throughout the depth of the filter.
Filtration characteristics are more easily defined for surface filters and a pore size can be specified, but depth filters offer the advantages of high flow rate and high capacity for collecting particles.
The most commonly used surface filters are membranes and, because of the accuracy with which the pore sizes can be defined, they are extensively used for studying microbes in hospital laboratories, in food and beverage QC, and in the sterility testing of pharmaceuticals and cosmetics.
Cellulosic membranes are the most widely used, especially for aqueous samples; while Cyclopore and Anopore inorganic membranes provide high filtration performance for demanding applications, or when microscopic examination of the collected particles is required.
Cyclopore track etched membranes are manufactured form pure polymeric films using patented Cyclotron technology (developed by the Catholic University of Louvain, Belgium) rather than the nuclear bombardment traditionally used for making track etched membranes.
Heavy ions
The tracks are created by beams of heavy ions which are subsequently chemically etched to give exceptionally accurate pore sizes with uniform distribution.
The result is surface capture with sharp and distinct particle size cut-off and a perpendicular pore structure with no lateral flow. They are available in pore sizes from 0.1 to 12µm.
This type of membrane is now widely used for cell culture, chemotaxis and cytological analysis by direct staining or isotopic assay. This is available in white or black, the latter introduced for researchers undertaking epifluorescent microscopy or other techniques where a contrasting background is advantageous.
Anopore, on the other hand, is composed of a high purity alumina matrix manufactured electrochemically. These membranes contain a densely packed array of regular pores running through them and they are virtually transparent when wet.
They are widely used as a support for cell culture, for encapsulation of pharmaceuticals in liposome manufacture and in biosensors (for controlled diffusion of analyte or as a matrix for incorporation of conductive materials).
As well as devices for vacuum and pressure filtration, Anopore and a variety of other micro- and ultrafiltration media are also available in microcentrifuge tube filters. Microcentrifugation is growing rapidly in popularity for filtering small sample volumes, and provides scientists with an additional and effective alternative for sample preparation, particularly when the use of ultrafiltration media is required.
In instances where the sample blocks a membrane filter too quickly, a glass microfibre prefilter should be used upstream of the membrane. Glass microfibre filters are depth filters that complement membranes, so will greatly extend the life of the experimental system.
Cell fragments
Moreover, their combination of properties renders them just as valuable for a wide range of biochemistry applications, especially for the collection of labelled protein or DNA precipitates and cell fragments.
High performance liquid chromatography (HPLC) is one of the most extensively used analytical techniques in pharmaceutical research, but thickening agents and tablet packaging can be particularly damaging to injection valves as well as causing rapid blockage of HPLC columns and tubing. Unfortunately these substances would equally quickly clog most conventional filters. An answer is to use something like a GD/X syringe filter. In addition to a wide choice of filtering membranes, these syringe filters include a prefiltration stack of graded density glass microfibre, which efficiently removes larger particles down to 0.7µm from the sample, leaving the final membrane filter to remove the fine particulate materials to the desired size. The result is that a three to seven times greater volume of sample can be filtered compared with an unprotected membrane.
Buffer solutions
Analytical buffer solutions should always be filtered as they are prone to the formation of precipitates and often support bacterial growth. They can be sterilised by filtration and stored in a reservoir with bacterial air vents.
For instance, a Polydisc ASTM with a 1µm pore filter could be used for filtering small batches of aqueous solvent, or with a 0.2µm pore size if the buffer needed to be sterilised. A Polycap AS capsule filter would sterilise large volumes of aqueous buffer.
Biotechnology applications pose a different set of demands to those of the analytical chemistry laboratory. Filters are used to produce clean and sterile gases for sample preparation areas and instruments.
Fermenters and bioreactors require a supply of sterile gases and there is an additional need for sterile venting. High flow rates of sterile gases at moderate pressure differentials can be achieved using glass microfibre media treated to be mildly hydrophobic for resistance to bacterial growth.
It is equally important to maintain sterile environments within cell culture vessels, and whilst cotton plugs in an Erlenmeyer flask containing growth media and cells have been the traditional method of choice for growing microbes, cotton is hydrophilic by nature so will become wet during autoclaving or in a high humidity environment and sterility is lost.
This can be avoided by covering vessels with a polymer-based cloth to ensure dry conditions, but this is a separate application step that increases both preparation time and labour.
Recently Whatman introduced the BugStopper to its line of sterile venting products. This is a reusable, sterile closure made of biosafe silicone containing a hydrophobic ultrafine glass microfibre filter. A stainless steel reinforcement ring surrounds the filter to provide support, and the entire assembly is simply pushed into or over the flask neck to provide a vent.
While air has free access, the closure retains 99.97 per cent of all particle of 0.3µm or greater in size and guarantees culture integrity. The device can be autoclaved in situ for total sterility, and is available in sizes for culture vessels of up to 2500ml capacity.
Nowadays, biotech research is becoming more and more automated, with different associated demands on the means of sample preparation.
Aiding batch filtration
Whatman has recently introduced a range of Filter Tubes for aiding batch filtration in combinatorial chemistry techniques. This range is manufactured to strict quality control standards for guaranteed reliable and reproducible performance under automated conditions, and is compatible with the Whatman SPE vacuum manifold as well as other well-proven automated equipment like the Gilson ASPEC system.
Filter Tubes feature a pigment-free polypropylene housing, chemically resistant to a wide range of solvents. Each unit comprises a housing, filter support and a choice of PTFE filter in pore sizes up to 5µm or phase separator filter paper. The filter is securely welded to ensure that it cannot be bypassed and precious sample lost.
Filtration microplates
Another very different range designed for automated robotic handling and centrifuge carriers is filtration microplates. These are manufactured in the standard multiwell format and have become widely used in automated biological screening applications and industrial scale research where there is a high sample throughput.
Whatman filtration microplates have a filter or membrane encapsulated in the base of each well and are produced using a patented process that ensures well to well isolation for zero cross talk.
They are available in a choice of materials to cater for a wide range of biological and organic sample types and storage applications and are available with matched collection plates and accessories.
Other, more specialised types of microplate include a protein precipitation plate, phase separation plate, PCR plates and glass and UV transparent-bottomed collection plates.
Changing requirements
Filters are developing to meet the changing requirements of life scientists. The choice of media is wide and, by incorporating a variety of high efficiency filters and membranes into disposable microfiltration devices, easy-to-use systems are available.
These devices offer excellent technical specifications and provide the combination of high performance and convenience so necessary in today's busy laboratory environment.
