Filtration Fundamentals: Is Knowledge Of Filter Technology Something You Let Fall Through The Cracks?

Date: September 28, 1998 Filtration Table An article on filters? Nah, filters are too simple. You put a small paper cone in the filter holder, add coffee, and pour on hot water. Drip, drip, drip, and the coffee's ready. That's all there is to it. But hold on a minute. There are thirty grades of Whatman standard filter paper alone, and even if we restrict ourselves to a biological laboratory, we have depth filters and screen filters; filters with different pore sizes that are tightly or loosely dispersed; packaging that employs prefilters, sheets, bags, tubes, or hollow fibers; filtration systems that use gravity, centrifugal force, vacuum, or pressure pumps; and systems that use a variety of different flow geometries (not even considering the closely related technology of binding assay membranes). Yet most people who simply use filtration as a "routine technique" in the laboratory give about as little thought to the process as they do to the decision to buy generic or brand-name coffee filters. Cross section of nitrocelluose membrane. Electron micrograph courtesy of Schleicher and Schuell Instead of looking at specific applications or examining the different syringe, bottle-top, centrifuge, capsule, cartridge, and myriad other filter holders, this article will outline some filtration basics and will focus on manufacturing and materials rather than applications and specialized holders, paying exclusive attention to the role of filters. Filtration can be loosely classified by the size or nature of the particles being removed from the liquid or gas being purified. Many companies break this spectrum down into five broad classes: macrofiltration, microfiltration, ultrafiltration, nanofiltration, and reverse osmosis. Macrofiltration is the coarsest category. Similar to every laboratory's core activity of removing coffee grounds from a flavored caffeine solution, macrofiltration generally involves removing particles larger than 1/20 millimeter. Sorting big rocks from small rocks at the local quarry would fall into the macrofiltration class, but for practical laboratory purposes we can accept an upper size range of a millimeter or so. For example, Whatman-type paper filter discs, as well as many different styles of glass, ceramic, and polymer prefilters, fall into this category. Microfiltration is at the smaller end of the particulate filtration spectrum. Microfiltration generally doesn't cover dissolved materials but will remove particulates between 0.2 µm and 10 µm. Most microfiltration filters are manufactured from polymeric materials, although some glass, ceramic, and even metal filters are available. Most filters fall into the microfiltration and ultrafiltration range, although a similar technology is used to produce filters for nanofiltration and reverse osmosis. Microrganisms are removed for fluid sterilization by this class of filters. Analytical test procedures are numerous using these filters. Ultrafiltration uses membranes to separate different organic macromolecules. An ultrafiltration membrane is generally semipermeable and requires considerable energy or force to effect the separation. Capable of concentrating bacteria, virus particles, and proteins, ultrafiltration is appropriate for molecules with molecular weights ranging from 1,000 to 1,000,000 Daltons. (Microfiltration, because it deals primarily with solids, is usually described in terms of particle size; ultrafiltration, because it can additionally be used to separate dissolved material, is generally described in terms of molecular weight.) Nanofiltration also uses membranes to separate different fluids or ions. The pores in a nanofiltration membrane are much smaller than those in an ultrafiltration membrane, so the energy required to drive the system is much greater. However, a nanofiltration process will eliminate most particles with molecular weights over 1,000 Daltons. In some ways, nanofiltration can be thought of as a process that lies somewhere between ultrafiltration (where the size and shape of a particle is responsible for its rejection by the filter) and the fifth broad category, reverse osmosis (where it is the charge on the particle that leads to separation at the ion level). Reverse osmosis effects the most difficult separations, employing a semipermeable membrane with the smallest pores and consuming the greatest amount of energy. Reverse osmosis will eliminate particles close to 100 Daltons. This means that the membrane will reject bacteria, viruses, most proteins, and many other small particles. However, the power of reverse osmosis comes from the fact that charge plays a far greater role in the separation than do size and shape, so much smaller particles are rejected by the charged membrane. Dissolved particles, if charged, will also be rejected by the membrane, even when the physical size of the dissolved material is considerably smaller than the pores. Because many common water contaminants carry a charge, such as salt, reverse osmosis is commonly used in water purification systems or desalination. PVDF membrane. Electron micrograph courtesy of Millipore The simplest membrane is the depth filter. Before looking at some of the materials used to manufacture filters, we should briefly examine the basic types of membranes. The paper funnel used to filter coffee is a form of depth filter; a depth filter can be thought of as a sheet or plug of material composed of randomly arrayed fibers or particles. The size of the particles or fibers, the density at which they are packed, and the means by which they are held together will influence the spaces between them. These spaces form a convoluted path that will allow passage of a gas or liquid as well as particles up to nominal defined size and shape. Depth filters are manufactured using three different approaches. For (often) crude applications, depth filters are manufactured by packing the filter material in a controlled fashion and bonding the particles/fibers appropriately. Perhaps the easiest to understand is the sintered glass filters that are commonly built into some kinds of glassware. Glass fibers or pellets are packed into a mold and subjected to heat and pressure. As the glass softens, it deforms and sticks together to create a "frit" of defined shape and thickness. The tortuous paths through the frit are controlled by the initial size of the particles and the amount of heat and pressure applied. This approach is taken for many glass and metal filters (when performed using metals, the product is usually referred to as molecularly bonded rather than sintered), but it is less commonly used with polymers. Silver metal membranes may be used directly for scanning electron microcopy (SAM) applications. They are also widely used for x-ray diffraction applications. Polymeric membrane filters are most commonly manufactured polymer filters or mats with a bonding material. The majority of membranes sold for microfiltration and ultrafiltration are manufactured using a controlled solvent evaporation process. Polymer is dissolved into an appropriate solvent. In a continuous process, a thin layer (often less than 0.2 mm) of the polymer solution is spread onto a moving belt and the solvent allowed to evaporate in a precisely controlled manner. As the solvent evaporates, channels and cavities form in the material, surrounded by blobs, fibers, and microscopic sheets of polymer. Surprisingly, this highly random process can result in membranes with absolute pore-size ratings. The majority of materials (including PVC, PTFE, nylon, and PES) used in polymeric filters are formed using this process. Because of limitations imposed by the properties of the material used, some polymeric filters are manufactured by the controlled stretching of a sheet of material. As the sheet is stretched, a combination of stretching and tearing leads to the creation of tortuous paths through the material. PTFE and polypropylene filters are manufactured using this process. More control is achieved by making precisely defined holes in an otherwise unbroken sheet of polymer, resulting in a membrane or screen filter. Such filters are manufactured using a process that gives control over the final product. A sheet of material is cast in an appropriate thickness. It is then passed over a source of charged particles that ionize paths through the material. Because ionizing radiation travels in straight lines, the damage tracks tend to be straight and at right angles, or close to right angles, to the surface of the sheet. Next, the damage tracks are etched away using a strong alkali solution, and finally, the alkali is neutralized and the filter washed. Precise control of the amount of exposure to the charged particles, the density of the charged particles that strike a defined area of the membrane, and the conditions of the etching process leads to membranes with well-defined densities of accurately formed pores. Isotropic membranes, including some track-etched polycarbonate and polyester membranes, have pores that are of constant diameter through the membrane, rather like short capillaries. As this process requires careful matching between the susceptibility to irreversible ionizing radiation damage and controlled etching, it cannot be applied to all kinds of materials. Polycarbonate and polyester are the most commonly used materials. Very few companies have the technology to perform this precise ion-etch process. Karin Dawson, at Osmonics, explained that ion etching (also called track etching) is a complex process to set up. "We started out as Poretics and later merged with Osmonics, which is why we have the technology base to manufacture the track-etch membranes," said Dawson. Poretics was founded by an engineer from General Electric who had originally pioneered the technique of track etching with Nuclepore, which is why the company has been one of the leaders in this field. Composite materials combine the desirable properties of multiple materials. In this sense, composite filters are created by sequential synthesis rather than simply mixing different chemicals. While a variety of approaches are used in their manufacture, most common is the controlled deposition of a material onto a preformed sheet or grid of support material. Composite materials are used when the physical features of one material (for example, the strength of the support material) need to be combined with the chemical features of another (for example, the high charge of the filter material). In addition to creating composite filters with desirable combinations of properties, improved separation power can be developed by layering materials. Conceptually, this is the simple idea of placing a coarse filter upstream of a finer one. An HPLC prefilter is a good example. Many companies now produce layered filters, where a coarse filter forms an integral part of the unit. The coarser prefilter first traps the "big junk" that would rapidly clog the finer filter, and the finer filter is therefore less likely to clog during the Before reviewing the properties of some of the filter materials, we should address a couple of other phenomena that are important and related to each other: clogging and fouling. Although there is considerable overlap, these processes can be considered to occur by very different mechanisms. In clogging, particles become physically wedged into the pores of the filter, either eliminating all further flow through that path or reducing the size of the channel. In fouling, particles and dissolved material bind to the matrix of the filter itself, narrowing the path and changing the filtration characteristic. Either of these processes will reduce flow through the filter and alter the nature of material that is retained. The effects of these phenomena can be reduced by choosing an appropriate flow geometry and filter type. If your concern is eliminating as much material from a liquid as possible (for example, removing cell wall and other insoluble debris from a cell extract), dead-end filtration is most commonly used in lab-scale purifications, for example, to sterilize heat-labile reagents. The solution is forced through the filter using gravity, vacuum, centrifugal force, or direct pressure from a pump or syringe. If there is a large amount of material to be removed in the solution, the filter will clog very rapidly. However, this old-fashioned approach is simple, fast, and inexpensive, so when the volume is small or the solution is already fairly clean, it is probably still the best method. Binding and fouling of the filter, which leads to a loss of filtrate, is probably less of an issue than guaranteeing that the filtrate you do collect is as clean as possible. If your concern is to eliminate as much of one kind of particle as possible while allowing passage of as much of another kind of particle (removing bacterial cells from a viral preparation, for example), a filter that will not bind the particle of interest is critical. Clogging or fouling of the filter will reduce the channel size so that the particle can no longer pass through. If the volume is large, fouling or clogging must be reduced as much as possible. Once a filter is fouled or clogged, the system must be stopped and the filter cleaned or replaced. The use of an inexpensive prefilter can alleviate some problems, and selection of an improved flow geometry can further reduce fouling and clogging. In these situations, cross-flow is preferable. In cross-flow filtration, the solution is flowed across the surface of the filter under pressure. Some solution passes through the filter, and the remainder continues to flow across the membrane. The cross-flow helps to eliminate some fouling (most notably particles that lodge in a pore on the surface of a screen membrane). Glass Microfibre Filters. Electron micrograph courtesy of Whatman Of course, all this discussion presupposes that you intend to use filtration to purify the gas or liquid being filtered. There are many other reasons to use filtration though. Screen membranes, for example, are often used to recover bacteria from solutions. After the solution has been filtered, the screen itself is used directly as a platform for microscopy. Polycarbonate screens, because they have almost optically smooth surfaces and do not absorb a variety of dyes and stains, are well suited for these techniques. Black membranes are also available to enhance contrast and facilitate fluorescence approaches, or if you'd rather culture what you capture, Schleicher and Schuell's NutraDisks"™ feature integrated filter and media pad combinations. Other filters are used to capture contaminants (environmental pollutants, for example), which are subsequently eluted from the filter or analyzed in situ. A lot to think about. And once you have decided on the type of filter that best suits your application, you'll need to consider what it should be made from: does the filter bind the material you are trying to collect, or will it swell or dissolve in the solvent system you are using? In fact, it's probably best to select the material first because some materials are more amenable to certain manufacturing techniques than others. While all this may sound like a very precise process, there is also some luck involved in selecting the perfect system. Osmonics' Karin Dawson explained, "The best place to begin is by ruling out filters that absolutely won't work for your application. Look at chemical compatibility. Does the membrane need to be hydrophilic or hydrophobic? Will you need to autoclave the membrane?" Once that process is complete, the list of possible membranes will be smaller. "At that point," she continued, "talk to some technical service representatives and try a few different products to see if they work. While one membrane might work for one person, it might not work as well for someone else. Lots of times it's a trial-and-error process that depends on individual equipment, techniques, and reagents." Here are some of the most commonly used materials; additional features and properties are described in Table 1. There are many different formulations of nylon, some of which are better suited for filters than others. Nylon is naturally hydrophilic, which is important in most biological applications, but it is also compatible with a wide variety of chemicals. Nylon is relatively robust, flexible, and stable in heat and cold. It is also fairly simple to add chemicals to the formulation to modify nylon's properties. Nylon membranes tend to contain very low levels of extractable materials, and are high protein binding, making them ideally suited for filtering sensitive analytical samples. Pall's nylon-66 has maintained a reputation for being the best. One such line is Micron Separations Inc/Osmonic's MAGNA nylon membranes, also manufactured from a nylon-66 polymer. Cellulose nitrate (nitrocellulose) filters seem to occupy a special niche in many researchers' minds, perhaps largely because of their extensive use in DNA and RNA hybridization procedures. Nitrocellulose is relatively inert, and as it is noncytotoxic, it is compatible with many biological applications, including work with cell lines. The downside in using nitrocellulose is that it is relatively brittle, making handling an issue for some applications. Large sheets (those used as transfer and hybridization membranes) can be strengthened by using the nitrocellulose as a coating on a stronger membrane base (such as nylon). Unmodified nitrocellulose filters can also be reinforced by careful design of holders. Nitrocellulose is used for filters but sees more extensive applications in hybridization membranes. Cellulose acetate membranes are generally low protein binding and high strength and are used more frequently than nitrocellulose in filters for filtration applications. Cellulose acetate itself is very inert, and several companies produce cellulose acetate or modified cellulose acetate membranes. A good combination of the binding characteristics of nitrocellulose and the strength of cellulose acetate is achieved through mixed polymer formulations. Whatman's WME (Whatman mixed ester: ~20 percent cellulose acetate and ~80 percent cellulose nitrate) and Osmonics' MCE (mixed cellulose ester) are good examples of these mixed formulations. Many dialysis techniques have been replaced by filtration, due to improvements in materials and the increased speed that results from the use of centrifuge or pressure-driven systems. Even in medicine, well-established techniques including kidney dialysis are being enhanced with filter-based approaches such as heme-filtration. However, modern dialysis techniques excel in the ultrasmall-volume niche, exemplified by Pierce's Slide-a-lyzer and Millipore's Drop-Dialysis systems, with sample volumes that are often smaller than 100 microliters. The membranes used in this type of application are usually some form of cellulose polymer, and the properties are better characterized than old-style dialysis tubing: Pierce's Slide-a-lyzer cassettes are available with membranes that have a range of molecular weight cutoffs (1 kD, 3 kD, 8 kD, 12 kD, 20 kD, and 50 kD--the company, Snakeskin dialysis tubing is a very similar material), while Millipore produces membranes with 0.05 and 0.025 µm pores. Polycarbonate is one material that is amenable to the ion-etching process described above. Polycarbonate is a clear, hard, durable material, which when combined with the precision achieved through the ion-etching poretics process leads to some versatile and useful filters. Osmonics/Poretics manufactures ion-etched polycarbonate filters (PCTE Polycarbonate Track Etched). The physical characteristics are also rigorously controlled, including thickness variation (+/- 5 percent), surface features, refractive index, autofluorescence, and a variety of filter and flow parameters. PCTE screens are available in a number of different formats with pore sizes ranging from 0.01 µm (6 x 10 8 pores per cm 2) to 14 µm (5 x 10 4 pores per cm 2). Only Osmonics/Poretics still use the original manufacturing method of exposing the polycarbon to the charged atoms of a nuclear reactor. www.osmonics.com/products/Page323.htm has photos of PCTE filter surfaces and also an interesting "cast" of the pores. PTFE membranes exhibit the widest range of chemical resistance, showing exceptional stability in strong acids, bases, and a large variety of solvents, including alcohols. PTFE is extremely hydrophobic, making prewetting processes a necessity for some applications, and it is also a weaker polymer than many of the materials discussed here. Some companies, such as MSI/Osmonics, laminate weak PTFE filters onto a polypropylene support, resulting in a filter that has polypropylene's durability coupled with PTFE's chemical resistance. While rather weak (often needing a supporting copolymer), polysulfone and the related polyethersulfone (PES) membranes are excellent for biological separations. These membranes tend to be nonpyrogenic, noncytotoxic, and low protein binding, making them ideal for filtrations involving cell lines and culture media. Polysulfone membranes are slightly tougher than PES membranes, but in general they shed more extractables. Low protein binding and low extractables make PES membranes an excellent choice for protein and nucleic acid purifications. While the membranes are fragile, prefabricated filter units (such as Millipore's Amicon units with which we are all familiar) allow for fast and efficient separations, concentrations, and buffer exchanges. PES membranes are available from many companies with an extensive selection of characteristics, rated by molecular weight cutoff or by pore size. MSI/Osmonics, in addition to its UltraSep polyethersulfone membranes, has a PES membrane that consists of a PES impregnated in a polyester support. These membranes have all t.he desirable characteristics of a polyethersulfone membrane but with the strength and flexibility derived from the polyester support. Again, these products are excellent for sterilization processes where low protein binding is important, such as tissue culture media sample preparation. Silver membrane. Image courtesy of Osmonics. The materials we described are the most commonly used polymers, but filters are also manufactured from many other materials. Glass and paper are often used as prefilters or in applications where high absorption or wicking properties are beneficial. Glass filters tend to be rather weak, but a number of chemical binders can improve their physical properties and modify their chemical characteristics. Ceramic filters often have desirable combinations of properties, but they can be very brittle; metal filters often overcome this limitation, but they can be expensive (however, ceramic and metal filters are often cleanable and reusable). Other polymers include the following: polyvinylidene difluoride (PVDF) filters are low protein binding, hydrophilic filters that tend to be physically robust but are available only in a limited range of pore sizes; polyolefin filters have excellent flow properties, but they are not autoclavable; acrylic filters tend to be extremely fragile, but they can frequently be reinforced with other materials. Company Web Address Micron Separations Inc. www.msifilters.com Pall Gelman www.pall.com Osmonics/Poretics www.osmonics.com Whatman www.whatman.com Millipore www.millipore.com Sartorius www.sartorius.com Schleicher and Schuell www.s-and-s.com www.s-und-s.de (in Europe) There are also other polymers, as well as laminates and mixtures. Filters are far more complex than might initially be imagined; in fact, it would probably take a book to adequately cover even the basics. But thankfully, many other resources are available, and most companies have excellent technical support. The "big players" are, for the most part, predictable names. Whatman, although competing with a number of other sources, is still the company most people turn to for paper filters. Pall's merger with Gelman Sciences expanded an already formidable array of technical expertise. Similarly, Micron Separations and Osmonics have merged with Poretics to form: "Osmonics Laboratory Products," so we can hope for a consolidated and improved product line in the near future. Finally, Schleicher and Schuell have a surprisingly extensive line of filtration products. In the United States, Schleicher and Schuell has a reputation as a hybridization membrane supplier, but in Europe, according to Schleicher and Schuell's Joseph Small, "We are more widely known for filters; people tend to underestimate the breadth of Schleicher and Schuell. We have an extensive line of filtration products that address the needs of many laboratories." Small said that the filtration details on the company's web site are currently only in German (at www.s-und-s.de on its European site) but that an English-language version is in the works. Schleicher and Schuell is clearly another company to consider when you need filters. When you start thinking about new filter applications, you should also consider checking out these companies' web sites. Much of the information in this article was collected from the web, and some companies (most notably Osmonics and Micron Separations Inc.) maintain huge web sites that contain plenty of valuable information. Some good starting points are in the table below. Bob Sinclair is a freelance writer based in Salt Lake City, Utah. He can be reached at bobsinclair@tech-write-edit.com. Filtration Table

Filtration Fundamentals: Is Knowledge Of Filter Technology Something You Let Fall Through The Cracks?

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September 1998

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