Still Spinning After All These Years: A Profile of the Ultracentrifuge

Date: October 11, 1999Ultracentrifuges Contacts for Ultracentrifuges Products Beckman Coulter's Optima MAX High-Capacity Personal Ultracentrifuge It boggles the mind when you think about it--a few pounds of well-balanced titanium or aluminum spinning steadily along at 1,667 revolutions per second, creating forces that approach one million times the pull of gravity. The ability to generate such speeds and forces through ultracentrifugation has contributed much to our understanding of biological sciences and has made ultracentrifugation a most basic and essential separation technique. While the physics of centrifugation never change, the machines and rotors that exploit those physical laws continue to evolve. The result is a growing collection of instruments for applying ultracentrifugation in new and exciting ways. In this profile, LabConsumer peeks inside the ultracentrifuge and the two companies that supply them, Beckman Coulter and Kendro Laboratory Products. Many will remember when most plasmid preparations were performed by banding plasmid DNA in cesium chloride gradients. A ubiquitous albeit messy technique in the 1970s and '80s, this application was largely replaced by the introduction and extensive use of plasmid DNA miniprep kits and column-based methods for large-scale DNA purification. The remaining applications for ultracentrifuges, however, were still a long way from being eliminated. Frank Richards, senior product planning manager for the Centrifuge Instrument Systems Development Center of Beckman Coulter, explains. "There has always been a core group of applications for the ultracentrifuge, particularly in cell biology. People will always be homogenizing something like rat liver and preparing cytosol fractions with 100,000 x g spins. The ultracentrifuge does this very cleanly. Nothing will substitute for that for a very long time." Raymond Kirton, marketing manager of academic and government services for Kendro Laboratory Products, concurs. "The base market for ultracentrifuges is still there. There was a reduction in market opportunity, but we are now seeing a slight increase as researchers move into genomics and gene therapy. While it's difficult to measure, we have noticed a resurgence in [the use of ultracentrifugation for] plasmid preparations and subcellular fractionations as researchers find they need highly purified samples and materials that kits cannot provide." Despite the work and thought involved in creating new centrifuges and rotors, Richards explains that it's very difficult to envision all the applications for a particular centrifuge when it is developed and released to the market. As a case in point, he relates, "When we produced the Airfuge™, we had few ideas of what people would use it for. We thought that perhaps researchers working with lipoproteins might have some applications. Now it's amazing what people have been able to do with it." For instance, the Airfuge has found extensive use in hospital emergency rooms for preparing serum samples for immediate analysis. The Airfuge is also used widely in the tomato product industry to rapidly clarify tomato paste for refractometry to determine sugar content. Overall, Richards summed up the ultracentrifuge application issue this way: "If the tool is there, people will find uses for it." To illustrate, Beckman Coulter has recently introduced the Optima™ MAX High-Capacity Personal Ultracentrifuge with its MLA-130 rotor that spins up to 130,000 rpm, breaking the 1,000,000 xg barrier--a first in the industry. Richards says, "It will be a year or so before a paper appears with a use for it, but that is no different than anything else." Of course, all of the principal techniques of ultracentrifugation, such as differential and density gradient separations, are well documented by Beckman Coulter and Kendro Laboratory Products. Both companies provide application assistance for use with a variety of samples and materials, including RNA, DNA, proteins, lipoproteins, viruses, and subcellular particles, in addition to excellent customer support and instrument field service and repair. Ultracentrifuges are useless without their rotors, so a considerable amount of energy goes into rotor design, testing, and production. At Beckman Coulter, for example, all rotors begin as an idea sparked by a research need. To test prototypes, Beckman runs them in their instruments at speeds high enough to bring about rotor destruction. This rigorous testing checks all of the components of a system, including the centrifuge. Once production begins, applications, training, and inspection programs are put into place. The list of available rotors is extensive and populated with details of tube sizes and numbers, volumes, maximum speeds, rcf values, and k factors. Briefly, most rotors are made of aluminum or titanium, but carbon fiber composite rotors (see sidebar) are beginning to find some application in the ultracentrifugation field as well. Five basic types of rotors exist. Swinging bucket rotors are used when maximum resolution of sample zones is needed. Fixed-angle rotors are useful for pelleting samples and isopycnic banding of DNA. As implied by the name, vertical tube rotors hold their tubes vertically within the rotor body, reducing path lengths and allowing fast run times for iso-pycnic and rate zonal separations. Beckman developed a variation of the vertical tube rotor, called the near-vertical tube rotor, for density gradient separations. When the tubes are tilted to an angle less than 10 degrees, pellets and floating materials are separated from the bands of interest. And lastly, continuous flow and zonal rotors have been designed for processing large sample volumes. As much as centrifuges require rotors, rotors require tubes. Beckman and Kendro have developed a variety of tubes, bottles, and tube sealing systems to optimize and simplify ultracentrifugation procedures. Kendro and Beckman take different approaches to rotor and ultracentrifuge combinations. Specifically, Kendro warrants its rotors to run in competitor ultracentrifuges and its ultracentrifuges to run competitor rotors. Beckman does neither. According to Beckman, the company tests its rotors and centrifuge combinations as systems, which is important for acquiring CE approval and providing maximum user safety. Users are advised to fully understand their instrument and rotor warranties before running rotors in competitor machines. A handful of companies have manufactured and sold ultracentrifuges since their introduction in the late 1940s. Here is a closer look at what the present suppliers have to offer. As the long-established industry leader, the name "Spinco" is synonymous with ultracentrifugation. Started as a garage-based operation in 1946, Spinco was the first company to manufacture ultracentrifuges, introducing an analytical machine in 1947. In 1949 Spinco introduced the Model L, the first preparative ultracentrifuge that boasted a maximum speed of 40,000 rpm. Beckman Coulter purchased Spinco in 1954, forming the basis of its Spinco centrifuge division. Now with over 50 years of experience, Beckman Coulter leads the way in centrifuge design and innovation and currently offers several state-of-the-art ultracentrifuges. Beckman centrifuges feature vacuum-encased induction drives that extend drive life by eliminating high-speed vacuum bushings; contain reliable, environmentally friendly thermoelectric cooling devices; and employ moisture-purging vacuum systems. All are imbalance tolerant, letting operators fill tubes by eye and handle up to 10 percent differences in tube weights. The Optima Series of Enhanced Ultracentrifuge Systems consists of tabletop and floor model machines. The Optima line of floor model ultracentrifuges includes three instruments. Portrayed as the workhorse of the three, the Optima LE-80K provides advanced capabilities for laboratory separation needs. It accommodates commonly used rotors with forces up to 602,000 x g and speeds up to 80,000 rpm. The Optima L-90K runs at speeds up to 90,000 rpm and 694,000 x g. In addition to accommodating the standard rotor types, the L-90K is designed to handle zonal and continuous flow rotors for large volume separations. Like the Optima LE-80K, the Optima L-90K is operated effortlessly through a solid-state panel that offers finger-touch control of run parameters, a choice of acceleration and deceleration profiles, and rapid recall of stored programs. Beckman Coulter's top-of-the-line ultracentrifuge, the Optima XL-100K, zips up to 100,000 rpm and produces a maximum force of 802,400 x g. The XL-100K is equipped to handle all of Beckman Coulter's ultracentrifuge rotors, including the new NVT 100 Rotor and the Type 100 Ti Rotor. In addition to its faster speed capabilities, the XL-100K is distinguished from the LE-80K and the L-90K by its advanced control and software systems. Its control panel reduces menu-driven operations for optimizing run times, separation efficiency, and system safety to a variety of keypad-based functions. In addition, the XL-100K performs a variety of calculations and conversions, such as duplicating runs and pelleting times in different rotors. Also found on board the XL-100K is the ESP™ Efficient Sedimentation Program. Developed through a collaborative effort between Beckman Coulter and the National Institutes of Health, the ESP graphically simulates plasmid DNA and RNA separations in ethidium bromide-CsCl gradients in real time. It then determines optimal run conditions for the specified rotor/tube/sample combination and stores these parameters in memory. Banding plasmid DNA in cesium chloride is one of the most commonly performed preparative ultracentrifuge techniques; therefore, the careful analysis of all the required parameters can dramatically improve time and separation efficiency. Beckman Coulter provides a line of at least 50 rotors for the Optima floor model ultracentrifuges. All five rotor types described earlier are represented, and maximum rotor capacities run from 16.5 ml (72x230 µl) to 1,500 ml (6x250 ml). Beckman Coulter has extensive information on rotor recommendations for key applications, such as separation of subcellular particles, viruses, and lipoproteins. Motivated in part by an observed trend toward smaller sample sizes and facilitated by small, solid-state thermoelectric cooling systems, Beckman Coulter inaugurated the personal ultracentrifuge market by introducing the Optima TL Personal Benchtop Ultracentrifuge. Designed originally as a temperature-controlled Airfuge, the TL evolved into a tabletop ultracentrifuge designed to run a series of baseball-size rotors with maximum total volume capacities spanning from 7 to 40.8 ml. At a top speed of 100,000 rpm, the Optima TL generated a force of 543,000 x g. The current version, the Optima TLX, has a top speed of 120,000 rpm and dishes out 627,000 x g. Responding to customer needs, Beckman Coulter raised the height on the Optima TLX and enlarged the chamber, creating the new Optima MAX Personal Benchtop Ultracentrifuge. The larger chamber of the Optima MAX spawned four new ML-series high-capacity, high-force rotors, thus extending the volume capacity of tabletop ultracentrifugation. In particular, the MLA-80 and the MLN-80 rotors have maximum sample capacities of 64 ml (8x8 ml), but most notable is the new MLA-130 rotor. Besides the four exclusive Optima MAX rotors, an additional 10 rotors run in all three tabletop machines. All are backed by a five-year warranty. Numerous benefits stem from the small size of tabletop machines. As a result of the shorter path lengths produced with smaller sample volumes, run times in the tabletop ultracentrifuges are significantly reduced. For example, virus separations are completed in one hour and membrane separations are finished in 15 minutes. And because of their lower profiles, the machines have lower centers of gravity, providing optimal stability. Sorvall's Discovery Series of ultracentrifuges Perhaps better known as Sorvall, Kendro Laboratory Products has roots in New York City. In 1934 Ivan Sorvall Inc. was founded there to import the newly invented angle centrifuge from Sweden. Ivan Sorvall entered the ultracentrifuge market with the OTD-2 in 1972. E.I. DuPont de Nemours eventually acquired Ivan Sorvall in 1973, and it became a valued part of the DuPont Medical Products Division. This relationship continued until 1996, when DuPont divested its Medical Products Division and Sorvall continued under the name of Sorvall Products LP. Finally, in 1997 Sorvall merged with a division of Heraeus Holdings to form the present-day Kendro Laboratory Products. As a result of a relationship with Hitachi Koki Co. Ltd., Kendro now offers three full-size ultracentrifuges in addition to two micro-ultracentrifuges. Known collectively as the Discovery™ Series, all five machines represent the latest in ultracentrifuge technology. Each contains an extremely reliable variable-frequency induction drive and features advanced thermoelectric cooling that eliminates CFCs and other ozone-depleting refrigerants. The Discovery M120 delivers 650,000 x g (with the S120-AT3 rotor) at a maximum speed of 120,000 rpm, while the M150 subjects samples to 900,000 x g (with the S150-AT rotor) at 150,000 rpm. Both machines feature safe, straightforward rotor loading and unloading and are simple to use via a "flip-back" LCD panel. An advanced damper bearing absorbs vibration, allowing visual balancing of samples and protecting both the samples and drives from damage. These durable drives are backed by a five-year warranty with no limit on the number of revolutions. A selection of 13 rotors, including vertical and swinging bucket configurations, is available and accommodates sample sizes ranging from 0.2 to 13.5 ml. Like the drives, each rotor is backed by a five-year warranty regardless of the number of runs or hours of use. The other three full-size ultracentrifuges are part of the Discovery Series. They have sample capacities up to 250 ml, run up to 100,000 rpm, and run zonal and continuous flow rotors in addition to the standard fixed-angle and swinging bucket rotors. Kendro offers the Discovery 90 and the Discovery 100 Ultracentrifuges that run at speeds up to 90,000 rpm (692,149 x g) and 100,000 rpm (802,006 x g) respectively; these workhorse machines share key features. Both feature easy entry of run parameters, quiet operation (<53 dBA), and sizes smaller than competitive floor models. Kendro's top-of-the-line model, the Discovery 100S Ultracentrifuge, offers all of the centrifugation capabilities of the Discovery 100, but adds a powerful software package, Compass™ Centrifugation Software. This package provides a state-of-the-art user interface that enables comprehensive setup, run simulations, advanced online calculations and conversions, online help, and run scheduling. In addition, an integral print function provides hard-copy verification of run parameters for GMP and other regulatory requirements. Kendro provides at least 35 rotors for use in the full-size Discovery ultracentrifuges. Maximum speeds range from 28,000 to 100,000 rpm. Individual tube sizes run from 2 to 250 ml, and the rotors hold from six to 40 samples. Because many labs have invested in rotors from other manufacturers or have specific rotor requirements, Kendro has designed and warrantied the full-size Ultra-Pro 80 and the Discovery floor model ultracentrifuges to run competitive rotors as well. Michael Brush can be contacted at mdbrush@compuserve.com. Nalge Nunc ultracentrifuge tubes Beckman Coulter and Kendro Laboratory Products are well known for the quality tubes, rotors, field service, and customer support they provide after a researcher purchases one of their ultracentrifuges. Other companies, however, have carved their own niches in the ultracentrifuge market by providing alternative sources for tubes, rotors, and instrument repairs. Two companies are the main alternative sources for ultracentrifuge tubes. Nalge Nunc International manufactures tubes and bottles from six different plastic resins compatible with a tremendous number of rotors. According to Liz Reagan, labware marketing manager for Nalge Nunc International, the best way to find tubes for a specific rotor is through the Centrifuge Ware Selection Guide found on the Nalge Nunc Web site at www.nalgenunc.com. The guide allows users to search through the Rotor Matching Database containing information on nearly 170,000 specific rotors, including many ultracentrifuge rotors. Seton Scientific of Los Gatos, Calif., was founded in 1983 to provide an alternative source of tubes, bottles, and accessories strictly for ultracentrifugation. In addition to carrying a complete line of open-top and sealed tubes, Seton manufactures tubes and tube sealers on an OEM basis. For additional information, contact Seton at (800) 543-2134. Perhaps one of the more exciting developments in rotor technology has been the composite, or carbon fiber, rotor. Carbon fiber, essentially the same material as that found in graphite golf clubs, tennis rackets, and the Stealth Fighter, has many benefits when applied to centrifuge rotors. Composite rotors cost less than their metallic counterparts, will not corrode, and are as much as 60 percent lighter than comparable titanium and aluminum rotors. Not only does the reduction in weight greatly improve their handling characteristics, composite rotors require shorter run times as well. The reason is simple--lighter rotors accelerate and decelerate faster than heavier metal ones. Composite rotors also retain their shape much better than metallic rotors and can be used for as long as 30 to 40 years without derating. Two companies are competing head-to-head in the carbon fiber/composite rotor market. Composite Rotor™ Inc., the original developer of the composite rotor, and FIBERLite Centrifuge Inc. differ in part by their rotor manufacturing processes. FIBERLite has developed a prototype centrifuge designed expressly for composite rotors. Called the EMD (Embedded Motor Drive) centrifuge, this unique device relies on the incorporation of a brushless DC electric motor embedded under the rotor. The stationary part of the motor, called the stator, is placed in the center so that the torque generated by the embedded motor spins the rotor directly. This arrangement eliminates the spindles, shafts, and couplers found on conventional drives and results in a 50 percent increase in motor efficiency. This, in turn, permits a reduction of the motor size, leading to less heat generation. In addition, the rotors are enlarged to make room for the motors, resulting in greater RCF values without increasing rotor speeds. Presently, composite rotors consist only of fixed-angle configurations, but swinging bucket-type rotors are on the horizon. While the majority of composite rotors are designed for high-speed applications, a few ultraspeed rotors are available. The National Independent Service Association (NISA) is an organization of 22 independent companies whose primary job is to provide in-house service for centrifuges and other scientific instruments. Scattered across the country, the NISA companies consist largely of former Beckman and Sorvall field service engineers. John Silva, a NISA member who started Orange, Calif.-based Southern California Scientific after 31 years with Beckman, says that "99 percent of the NISA members are factory trained, and most have many, many years of experience." Indeed, the four employees of Silva's company have over 100 years of collective field service experience with centrifuges, spectrophotometers, and scintillation counters. Silva explains that as independents, the members of NISA offer high-quality work and fast response times at lower hourly rates with reduced prices for service contracts and replacement parts as well. In addition, they will repair and rebuild instruments and components that the original manufacturers will no longer service. Ultracentrifuges Contacts for Ultracentrifuges Products

Still Spinning After All These Years: A Profile of the Ultracentrifuge

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