Manufacturers Respond To Scientists' Need To Speed Filtration Process

For researchers who made solutions a quarter-century ago, filtration involved deftly folding a round piece of filter paper into a triangular shape so that it would fit inside the inverted cone of a funnel. Once the paper was fitted inside the funnel, they could filter practically any solution, even those thick with solute--providing they had hours of time to spend watching the clear liquid drip into a flask as the particulate material in the funnel grew thicker and thicker. If they were in a real hurry, they could add a vacuum pump to the filter and flask, to pull the particle-free solution through the filter faster. There were limitations to such filtration systems, though, such as trying to remove the rubber stopper holding the funnel from the flask, after the vacuum had pulled it tightly into the neck. While filter paper and funnels will always have a place in chemistry labs, over the last decade filters made of materials such as polysulfone, polytetrafluoroethylene (PTFE), and cellulose acetate--and systems that speed the filtration process--have all but replaced the paper rounds in the laboratories of life scientists. For example, using membrane filters in vacuum filtration systems, microbiologists can collect and analyze microbes in samples of food and water, and environmental engineers can assess air quality in buildings or outside. Cell biologists sterilize culture media by passing their delicate mixtures of amino acids and vitamins through filter membranes made of cellulose acetate and cellulose nitrate, rather than risk destroying the medium components by heat sterilization. Valuable laboratory mice at breeding farms and in university research facilities breathe nothing but the highest-quality air, purified by membranes that filter out particles even smaller than a micron. Perhaps most important is the laboratory-grade water used by countless biologists and chemists, who require for their experiments highly purified water prepared by any one of several filtration techniques. "For most research applications, the water should be free from all types of contaminants," says Mark Brolaski, president and laboratory director of MoBio Laboratories, an independent product testing laboratory in San Diego. "This is especially true when the water will be used in molecular biology applications, particularly those involving RNA, and in cell culture, where water is required for culture medium preparation." Practically every laboratory requires copious amounts of water--for making buffers, for instance, or as a solvent for biological substances such as proteins and DNA. Preparing laboratory or reagent-grade water often demands several purification steps to remove contaminating ions (sodium, chlorine, and calcium, among others), dissolved ionized gases (carbon dioxide), organic chemicals both natural and synthetic, and microorganisms or other particles that may have found their way into a water supply. The two oldest methods for producing purified water are filtration and distillation. "Distilled water is produced when water is heated to its gaseous state and then allowed to recondense," explains Rick Passinisi, product manager at Barnstead/Thermolyne Corp. in Dubuque, Iowa, a company specializing in water-purification products. "Many of the impurities commonly found in water--including dissolved salts, sand, or dirt, and microbiological agents--are left behind as the water vapor condenses." Far from the classic metal Barnstead stills, first developed more than a century ago by chemist Robert Barnstead, current distillation systems such as the Mega-Pure system by Barnstead are compact, computer-controlled systems that purify and collect water automatically. For labs that lack space, Barnstead's smaller FI-Streem II glass stills take up less than 2 feet of bench space and still put out 4 or 8 liters of distilled water per hour. Those partial to the old workhorses will be glad to know that Barnstead continues to manufacture its steam and electric Classic stills. Distillation, however, does not always remove dissolved ionized and organic gases--the most common of which are oxygen and carbon dioxide--from water. For analytical procedures such as high-performance liquid chromatography (HPLC) and atomic absorption spectrophotometry (AA), in which even trace amounts of these substances can lead to errant test results, the laboratory water must be absolutely free of organic and ionic impurities. Producing reagent-grade water suitable for applications like these can be accomplished by several filtration technologies. These methods include deionization, filtration and ultrafiltration, reverse osmosis, adsorption of impurities to activated carbon, and pretreatment with ultraviolet (UV) radiation, which oxidizes dissolved organics in the water and facilitates their removal. Deionization, ultrafiltration, and reverse osmosis all involve forms of filtration for the removal of ions and gases from water. Deionization uses a charged resin to which oppositely charged compounds stick as water is passed through, a method known as ion exchange. In ultrafiltration, water flows past a membrane made of a material such as polysulfone or cellulose acetate that allows pure water through while holding back particles above a specified molecular weight, including microorganisms and some types of dissolved organics. As part of a water-purification scheme, ultrafiltration is used primarily to generate water that is free of pyrogens, which are bacterial toxins made of lipopolysaccharide or protein. Ultrafiltration membranes also have several other uses in the life sciences laboratory, including concentrating dilute solutions and purifying specific chemicals (antibodies, for example) away from larger (or smaller) substances found in the same solution. Reverse osmosis is considered a highly effective method to remove most of water's impurities. "Reverse osmosis removes 95 percent of ionized impurities and 99 percent of organic substance, microbials and other particles, and pyrogens [bacterially derived toxins]," states Passinisi. "The removal capabilities are broad, but reverse osmosis alone does not do a complete purification job. Most laboratories, therefore, use reverse osmosis as a pretreatment for additional filtration steps such as deionization." In natural osmosis, water diffuses across a semi-permeable membrane, from low solute concentration to high, in an effort to equalize concentration differences. In reverse osmosis, pressure is applied to the side of the membrane with a higher concentration of dissolved solids, causing the water to travel from high concentration to low, opposite the natural process. This way, ions and particles are concentrated and removed from the water passing through the membrane, reducing the load on the filters next in line. This can extend the life of deionization cartridges or ultrafilters used later in the purification scheme. Reverse osmosis and deionization are often combined with adsorption, ultrafiltration, and UV oxidation in systems that produce water containing nothing more than. With a system of cartridges containing resins or filters, the Milli-Q water systems by Millipore Corp. of Bedford, Mass., combine several methods of water purification--such as reverse osmosis, deionization, and ultrafiltration--into one system. Barnstead's EASYpure UV/UF system utilizes adsorption, deionization, filtration, UV oxidation, and ultrafiltration to create water that the company claims is free of practically all contaminants, including RNAses (RNA-degrading enzymes) and pyrogens. The water coming from the system is pure enough to be used in media preparation or in molecular biology experiments involving RNA. "RNAses and pyrogens are difficult to remove from water because they are not destroyed by sterilization techniques, such as autoclaving and gamma irradiation," says Brolaski. "Our laboratory has shown that the EASYpure UV/UF systems removes RNAses from source water without additional pretreatment steps," such as reverse osmosis. In many research instances, the purity of the air is as important as the quality of the water. Valuable transgenic mice, for example, with genes mimicking human disease genes or carrying parts of foreign genomes, are maintained in animal facilities in an exquisite state of health. Their food, the water they drink, and the air they breathe are all purified to prevent the passage of disease-causing microorganisms from the environment to the animals (H. Ahern, The Scientist, July 10, 1995, page 18). Cages that hold these animals are equipped with special filters that provide high-efficiency particulate air (HEPA) into animal housing systems. HEPA filters, such as the HEPA-grade filters made of a glass microfiber manufactured by Whatman Inc. of Clifton, N.J., prevent particles in air larger than 0.3 micron from passing through to the mice. Filters that permit bidirectional flow of gases allow only the "clean" air through while still allowing gases such as CO2 to flow out of the cage. "The animal cages with a wire lid [are] old technology now," notes Michael Hayre, director of the laboratory research center at Rockefeller University, referring to the days in which a laboratory animal simply breathed the air coming through a cage's metal cover. "The air that laboratory animals breathe is filtered through HEPA filters to keep airborne contaminants, which could possibly make an animal sick, out of the animal's environment." "HEPA filters retain particles larger than 0.3 microns at an efficiency of 99.97 percent," says Cheryl Lundgren, technical services representative for Whatman LabSales Inc., a subsidiary of Whatman Inc. in Hillsboro, Ore. "When you consider the pore size and the efficiency with which particles are retained, that's something to be excited about." When added to tubing carrying oxygen or carbon dioxide to incubators used to grow cells in culture, in-line filters such as the HEPA-CAP and HEPA-VENT by Whatman Inc. and the AcroVent filters manufactured by Gelman Sciences of Ann Arbor, Mich., prevent contaminants such as bacteria that might be present in gas supply lines or cylinder tanks from reaching the cultures in the incubator. Process-scale laboratories making pharmaceuticals or research biochemicals use in-line filters--such as Whatman's Poly-Vent filters made of PTFE and housed in a square polypropylene capsules--to protect the contents of collection vessels from contamination by outside sources, and also to protect the laboratory from the vessel contents. Life scientists have many other uses for filters, which usually involve purifying, concentrating, or separating a substance such as an antibody or an enzyme from biological fluids. Consider a cell biologist who has carefully cultured cells expressing an important protein or peptide--perhaps an antibody or a cytokine. The cells in the culture vessel are bathed in culture medium rich in amino acids; vitamins; and, in many cases, serum, a compendium of proteins, salts and water. Since the antibody is secreted by the cultured cells into the culture medium, how might the cell biologist separate the antibody of interest from the myriad of other proteins in this biological mixture? "The best method is ultrafiltration," says Lundgren of Whatman LabSales. Ultrafiltration separates extremely small particles and dissolved molecules from biological fluids on the basis of their molecular size. Substances smaller than the pore size of an ultrafiltration membrane, which can range from 1,000 to 1 million molecular weight, pass through the filter while larger ones are held back. Either portion, containing the small molecules or the larger ones, can be collected. Although there are several types of ultrafiltration membranes available, those gaining increased use in laboratory-scale and process-scale applications are hollow-fiber and spiral-wound ultrafilters and cassette cartridges, in which the membrane is held in a cassette resembling a large projector slide. Hollow-fiber ultrafilters consist of cylindrical cartridges packed with long hollow fibers, made typically of polysulfone, through which the liquid to be filtered flows. As molecules pass through each fiber membrane's core, substances smaller than the molecular cutoff traverse the membrane, while those that are larger than the pores are progressively concentrated. Spiral-wound filters similarly contain layers of filter material coiled around a permeable collection tube at the center of the cartridge. Again, molecules larger than the pore size are retained and collected. The advantage of both of these systems is the size of the filter's surface area, which far exceeds the surface area of most types of membrane filters. These types of membrane cartridges are well suited for processing biological solutions containing mixtures of macromolecules or suspensions of cells. To maximize the life of the ultrafilters, ultrafiltration is often performed in a tangential flow mode, which means that the fluid being filtered flows over the surface of the filter as well as through it. "Tangential flow systems impart a transmembrane pressure so that fluid flows across the membrane and also through it," explains Cheryl Rich, technical services representative at Pall/Filtron Technology Corp. in Northborough, Mass. This keeps the filter from becoming clogged with particles larger than the molecular size cut-off. Microporous membranes, which have pores larger than those of the ultrafilters, are also amenable to tangential flow. Microporous membranes are used during cell-harvesting procedures and for concentrating particles such as viruses. Amicon Inc. of Beverly, Mass., has several systems incorporating hollow-fiber, spiral-wound, or cassette ultrafilter membranes, ranging in size from benchtop systems for laboratory-scale processing (some with tangential flow capabilities) to much larger systems, such as the ProFlux systems, which are designed to process thousands of liters for production-scale applications. Millipore's Minitan and Pelicon Filter systems, with cassette-type ultrafilter membranes, are used to concentrate biochemicals and biopharmaceuticals, such as monoclonal antibodies, enzymes, and cytokines from suspensions of cells or cell-culture supernatant. Pall/Filtron also carries a range of ultrafilters and systems that are used in the pharmaceutical, biotechnology, and food industries for concentration or fractionation of biological fluids. Separating, sterilizing, or purifying biological liquids in amounts significantly smaller than the kiloliter range can be accomplished using filters packed into syringes and centrifuge tubes. Because most cell-culture media are heat-sensitive, they are sterilized by filtration rather than autoclaving. Corning Costar Corp. of Cambridge, Mass., sells several types of filters that allow the person preparing a sterile liquid to directly filter the solution into a presterilized bottle or collection tube. Along with Corning Costar, Pittsburgh-based Fisher Scientific, Sigma-Aldrich Techware of St. Louis, and Alltech Associates of Deerfield, Ill., all carry filter cartridges that fit on the end of a syringe. Fisher offers a variety of syringe filters from the product lines of Whatman, Gelman, and Corning, along with Fisherbrand dispensers, which hold up to 150 syringe filters. Sigma-Aldrich Techware carries disposable units in an array of molecular cutoffs and sizes. SYTRINGE FILTERS: Alltech Associates' Novo-Clean IC Disks remove both particles and dissolved contaminants from separation solutions. Syringe filters can be used for sterilizing small amounts of a liquid such as cell-culture solutions of antibiotics or amino acids used for cell culture. Some types of syringe filters, including the Ion Chromatography Acrodisc from Gelman Sciences and Novo-Clean IC disks manufactured by Alltech Associates, remove both particles and dissolved contaminants from solutions bound for an analytical separation procedure like HPLC. The so-called spin filters, which are usually ultrafiltration membranes contained within a centrifuge tube, have many different uses in the biology laboratory, including separating, concentrating, and desalting biological solutions. With the Ultrafree-MC filter units from Millipore, for example, microliter volumes are filtered through ultrafiltration or microporous membranes, purifying minute amounts of nucleic acids or enzymes or concentrating viruses. The Ultrafree-CL filter units process larger volumes (in the milliliter range) and can be used to concentrate proteins before gel electrophoresis or to purify DNA or RNA products. SPIN DOCTORS: Amicon Inc.'s Centricon concentrator, left, for antibody concentration, removal of primers from amplified DNA, and removal of nonincorporated linkers and labels. Right, the company's Microcon microconcentrator, for removing nonincorporated primers from PCR-amplified DNA. Centrifugal concentrators--such as Amicon's Microcon, Centricon, and Centriplus microseparation tubes and the NanoSpin and Z-Spin centrifugal devices from Gelman Sciences--allow researchers to concentrate or desalt solutions of proteins and nucleic acids following preparative procedures, including column chromatography and HPLC. The steps are simple--a solution is placed into the centrifuge tube containing the miniaturized ultrafilter and spun in a microcentrifuge or an ultracentrifuge, depending on the sample size. When the centrifuge stops, the separation is complete. FOR SOLUTE RECOVERY: The Centriplus concentrator from Amicon for samples of up to 15 ml volume. Environmental applications--for example, detecting microbes in water and food, or analyzing air samples for airborne asbestos--also include filtration steps. Microbiologists monitor drinking water supplies for coliform bacteria by vacuum-filtering samples through membrane filters of cellulose acetate and cellulose nitrate (mixed esters), which retain particles larger than a few microns. The filters are cultured, and the bacteria grow into colonies that can be counted to provide an estimate of the quantity of bacteria in the supply water. Testing can be performed in the laboratory or in the field. Millipore's Portable Water Laboratory is a self-contained kit that provides filters and microbiological supplies, including media and a portable incubator, to detect and identify coliform bacteria at the water's edge. Air can be analyzed for particulate contaminants, such as nuisance dust or asbestos, in a similar way with an air-test kit. Particles in an air sample are left behind on a filter as the air is sucked through a membrane. A microscopic analysis of the particles on the filter reveals the presence of particulate membrane in the air. Membranes for air monitoring are available in both white and black, so that both dark and light particles are highly visible to the investigator. Faced with so many choices of filters and filtration techniques, researchers must carefully consider their options when selecting a filter for a particular application. "Choosing a filter material is not simply a function of pore size and particle size," comments Lundgren of Whatman LabSales. "The nature of the analyte and the type of solvent are also important considerations. The goal is that the analyte should be soluble in the solvent, but the membrane shouldn't." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y. (The Scientist, Vol:9, #17, pg.18-19 , September 4, 1995) (Copyright © The Scientist, Inc.)

Manufacturers Respond To Scientists' Need To Speed Filtration Process

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