Principles of Continuous Flow Centrifugation

Content Type: Technical Application Note
Authors:
Mel Dorin
Judy Cummings
Beckman Coulter, Inc., Indianapolis, IN 46268

To process material in conventional centrifugation rotors, the following steps must be performed:

  • Load the rotor with sample.
  • Accelerate to operating speed.
  • Run for a specific period of time.
  • Decelerate to a stop.
  • Unload the sample.

These steps must be repeated until the entire sample is processed. If the quantity of material to be processed is large, its sedimentation rate low, the acceleration/ deceleration time of the rotor long, and the rotor capacity small, conventional processing of large volumes of material will be labor-intensive and very time-consuming.

Continuous flow centrifugation is a laboratory time-saver, whereby large volumes of material can be centrifuged at high centrifugal forces without the tedium of filling and decanting a lot of centrifuge tubes, or frequently starting and stopping the rotor. For example, 10 L of liquid containing 500 S particles can be processed by continuous flow methods in 4 hours or less, depending on the rotor selected. [Note: The sedimentation coefficient is usually expressed in Svedberg units, where 1 S = 1 × 10–13 seconds. Thus, the sedimentation coefficient of a particle measured at 500 × 10–13 seconds is said to have a value of 500 S. The velocity of a particle in a centrifugal field can be defined as s = v/ωr2, where s is the sedimentation coefficient, v is the velocity of the particle in centimeters per second, ω is the angular velocity of the rotor in radians per second, and r is the distance from the axis of rotation in centimeters. Sedimentation coefficients, as used here, are sobs (values observed for the particular solvent system and temperature used) rather than s20,w values for water at 20°C.] This same operation would require 12–21 hours by conventional batch-type centrifugation. This combination of high centrifugal force and high throughput makes continuous flow processing particularly useful for: the large-scale collection of viruses (either for research purposes or for the preparation of commercial vaccines); the sedimentation of bacteria; and the pelleting of subcellular fractions. However, continuous flow centrifugation need not be confined to biomedical applications. It should be considered whenever particles of any kind with sedimentation coefficients of 50 S or larger must be routinely separated from fluid volumes of 2 L or more.

Continuous flow rotors substantially minimize material processing time for 2 reasons:

  1. They have short pathlengths to reduce overall pelleting time. Hence, they efficiently pellet solids out of a sample stream and facilitate a rapid flow of material through the rotor.
  2. They have large capacities. Therefore, they do not need to be star ted and stopped as often as conventional rotors. This saves time by reducing sample handling and reducing the time lost in waiting for rotor acceleration/deceleration between runs.

Qualifying the Sample

Continuous flow rotors are of greatest benefit over conventional rotors when the sample has the following properties:

  1. The sedimentation coefficient of the particles to be collected is greater than 50 S. Because the rotor has high pelleting efficiency, solid material can be separated from the liquid medium faster than with a swinging bucket or fixed-angle rotor.
  2. The sample solid/liquid ratio is low (5–15%). Above a solid/liquid ratio of 15%, the rotor tends to be overefficient, i.e., it pellets so quickly that it fills immediately. This means that it must be stopped for unloading the pellets so often that too much time is lost in accelerating/decelerating the rotor and cleaning it between runs. Conversely, if the sample contains little solid material, the rotor will operate for long periods of time, processing large volumes of material between shutdowns.
Figure 1
Figure 1: Cross-section of a continuous flow rotor.
Figure 2
Figure 2: Continuous flow centrifugation. The arrows on the diagram indicate the direction of liquid flow during continuous flow operation. The sample is pumped in through the center inlet to the bottom of the core. The flow rate is adjusted so that the particles of interest have time to become trapped in the gradient or cushion (or pelleted on the rotor wall) during the time required to move from the bottom of the core to the upper radial channel.

Continuous Flow Operation

Both Beckman Coulter continuous flow rotors—the CF-32 Ti and the JCF-Z—operate on a continuous flow, batch-processing basis. That is to say, a batch of particlecontaining liquid flows continuously into the rotor, which is running at the selected operating speed. Particles sediment out of the flowing stream that then emerges as a particle-depleted effluent. This process continues until the particle-containing capacity of the rotor is reached, or until the starting material (sample) is completely processed. Thus, the amount of starting material that can be handled in one run is governed by the concentration of the particles it contains, as well as its volume. The sediment, the particle-depleted effluent, or both can be collected, just as one collects the sediment and/or supernatant after differential centrifugation in conventional fixed-angle or swinging bucket rotors.

Both the CF-32 Ti and JCF-Z rotors consist of a bowl, a solid core that is placed inside the bowl, and a lid (see Figure 1). Radial channels for the flow of liquids pass through the core. There are 4 milled slots on the top surface of the core through which liquids flow for loading and sometimes unloading the rotor. The continuous flow mode of operation is made possible by the rotating seal assemblies that permit the fluidbearing lines to remain attached to the rotors during operation. These seals are designed to prevent exposure of the sample to environmental air—a feature that minimizes possible contamination of the sample. For loading and unloading, the flow of fluid through the rotor can be reversed by simply switching the fluid lines.

The rotor is prepared for centrifugation while spinning at low speed. Buffer solution, cushion, or discontinuous layers of a gradient material, such as sucrose, are pumped in through the edge line. Then the rotor is accelerated to operating speed, the fluid lines are switched, and the particle-containing liquid is routed through the center inlet of the seal with the same pump. It is delivered through the channels in the rotor core to the bottom of the rotor bowl. This direct, sealed pathway virtually eliminates undesirable foaming (see Figure 2). As the flowing liquid moves upward along the core taper toward the top of the bowl, sedimentation of particles toward the bowl wall takes place. The particle-depleted effluent travels through the upper radial channel and leaves the rotor via the outlet (edge) line in the seal assembly.

Separation Techniques

Particles may be concentrated in one of 3 ways: by pelleting onto the wall of the rotor bowl; by sedimentation onto a cushion of dense liquid, such as sucrose; or by banding in a gradient. The first 2 methods are most useful for either harvesting the particles, or for recovering the particle-depleted effluent. Whenever it is necessary to separate a particle from contaminants of different densities, banding will give the best results.

Pelleting and Clarifying

Pelleting is suitable for collecting particles that won’t be damaged by being pressed against the wall of the rotor. It is the fastest continuous flow method and can handle large volumes of starting material. While operating at low speed, the rotor is filled with buffer

solution or starting material. Then the rotor is accelerated to the selected run speed, and starting material is pumped through the center line of the seal. The run is continued until all the material has been processed, or the maximum pellet capacity of the rotor has been reached. (The latter case may be apparent when the effluent emerging from the rotor becomes turbid.) At this point, rotor speed is maintained long enough for all sedimenting particles to reach the rotor wall, while buffer solution or water is pumped through the center line. Then the rotor is decelerated to rest, the supernatant remaining in the rotor is decanted, and the pelleted material is scraped off the rotor wall. Sometimes, two stages of flow-through centrifugation can be employed—a first-stage clarification at low rotor speed to remove unwanted large particles, followed by a second pass of the effluent through the rotor running at high speed to collect small particles. A virus-containing culture fluid, for example, may be cleared of cellular debris in this manner. The clarified effluent can then be reprocessed by pelleting or banding to concentrate the virus particles.

Sedimenting onto a Cushion

Particles that might lose biological activity if pelleted (some viruses, for example) can be sedimented onto a cushion of a dense solution such as sucrose. A cushion should be used whenever it seems more convenient to collect the particles in solution rather than having to scrape a pellet off the rotor wall. The particle-bearing capacity of the rotor will, of course, be reduced by the presence of a cushion.

For this type of run, the rotor is loaded while operating at low speed (see Figure 3). Buffer or water is introduced first through the edge line and over the milled slots on top of the core. Then the cushion (a 60% by weight sucrose solution, for example) is loaded in the same manner. It enters at the periphery of the rotor where it is held against the wall by the centrifugal force field, because it is more dense than the water or buffer. At this point, flow lines are switched, and buffer is pumped through the center line as the rotor is accelerated to operating speed. Once operating speed is reached, starting material is pumped through the center line. When sedimentation is completed, unloading (like loading) is done with the rotor turning at low speed. Air is introduced through the edge line to block the upper radial channels as shown in Figure 4. (This prevents displacement fluid from entering these channels and disturbing the particle-laden cushion during the unloading process.) Then the rotor is unloaded by pumping a dense solution through the edge line and over the milled slots, thereby displacing the rotor contents through the center line.

Figure 3
Figure 3: Loading a cushion or gradient. The arrows indicate the direction of liquid flow during loading. With the rotor turning at low speed, the cushion or gradient (light end first) is pumped in through the edge line. Air is displaced through the center inlet. The cushion or gradient is held against the rotor wall by centrifugal force.
Figure 4
Figure 4: Unloading a cushion or gradient. When the particles of interest are contained in a cushion or gradient, it is necessary to unload without mixing the rotor contents. With the rotor turning at low speed, air is introduced through the edge line to form a bubble blocking the upper radial channels. A dense solution is then pumped in through the edge line, forcing the cushion or gradient out through the center.
Banding in a Gradient

 

This method is most commonly used for the purification of viruses. Because of the short sedimentation pathlength in the continuous flow rotors (about 10 mm), it is not necessary to use a preformed linear gradient. Step concentrations will diffuse enough to become linear during the loading and acceleration process. Usually, 2 or 3 concentration steps of a gradient material, such as sucrose solution, are selected that encompass the density of the particles to be banded. The last and most dense solution acts as a cushion to prevent sedimentation to the rotor wall. The short pathlength makes it possible to band particles quite rapidly. However, depending on the buoyant densities of the particles, it may be difficult to completely resolve and collect multiple bands from within such a short gradient.

Operation is quite similar to that described under Sedimenting onto a Cushion. While the rotor is operating at low speed, fluids are loaded through the edge line in this order: buffer solution or water, followed by the step gradient (light end first), and the cushion last. The remaining sequence of operation is the same as described in the preceding section. In this case, however, sedimentation may be allowed to continue as long as necessary for banding of the particles.

Selecting a Flow Rate

The sedimentation coefficient of the particles to be collected governs the selection of flow rate and operating speed. Small particles require either a higher centrifugal force field, or more time to sediment than larger ones. The aim is to select an operating speed that will generate a centrifugal force high enough to sediment the particles of interest, and a flow rate low enough to provide time for these particles to sediment out of the flowing stream before it leaves the rotor. For most efficient operation, one tries to use a flow rate as close as possible to the theoretical maximum. (Nomograms that portray the relationship between theoretical maximum flow rate and rotor speed for particles of known sedimentation coefficients can be found on pages 11 and 12.)

Method A

The nomograms have been generated from the following equations, which may be used for determining an approximate flow rate, F, or rotor speed for specific samples.

Equation 1

F is expressed in mL/min. For the JCF-Z Standard Pellet Core, rt = 7.6 cm, and rb = 7.1 cm, reducing ri to 7.35 cm and Equation (1) to Equation (2a):

Equation 2a

For the Large Pellet Core, rt = 5.6 cm and rb = 5.1 cm. Thus, ri becomes 5.35 cm and Equation (1) reduces to Equation (2b):

Equation 2b

Note: The above equations assume that the density and viscosity of the liquid in which the particles are suspended are similar to water at 20ºC. If this is not so, an adjusted sedimentation coefficient, sr, should be calculated.

Before the nomogram or Equation (2) can be used, however, sr must be calculated from the sedimentation coefficient or the diameter, D, of the particle of interest. If the diameter is known, use

Equation 3

If the sedimentation coefficient (i.e., s20,w) of the particle is known, it may be used to calculate sr as follows:

Equation 4

Method B
A simple way to determine the flow rate for a continuous flow rotor is to use known k-factors to compute pelleting times from another rotor. The k-factor is a constant that is different for each rotor and is a measure of pelleting efficiency. It is derived from the equation:

Equation 5

For example, if separation is performed in a JA-10 rotor, and the time to pellet in a full bottle run at full speed is 5 minutes, then this information can be used to determine the pelleting time in the JCF-Z rotor in the following way:

Equation 6

Substituting the experimental values results in the following equation:

Equation 7

or t2 = 24 seconds to pellet in the JCF-Z rotor. If the volume of the JCF-Z rotor is 1000 mL, then we know we can pellet 1000 mL in 24 seconds, or we can use a flow rate of 41 mL/s (2.4 L/min). If the solid/liquid ratio of the sample is 5%, we can process 200 L of material (total time = 1:24 hours) before shutting down the rotor for cleaning. Comparing the 84-minute processing time to a JA-10 rotor, the JA-10 can process 1.5 L in 5 minutes; the time to process 200 L is 11:10 hours. The continuous flow rotor is at least 8 times faster than a conventional large-volume rotor such as the JA-10.

Zonal Core JCF Z Rotor

Zonal Core JCF Z Rotor

Figure 5: JCF-Z continuous flow rotor.

Rotors, Cores, and Their Uses

There are two continuous flow rotors currently available from Beckman Coulter: the JCF-Z rotor and the CF-32 Ti rotor. The JCF-Z rotor has a maximum speed of 20,000 rpm and is designed for use in select Avanti series centrifuges. It has 3 interchangeable cores for continuous flow operation and 2 for zonal runs. These are: the Standard Core; the Large Pellet Core; the Small Pellet Core; the Reorienting Gradient Core; and the Zonal Core, respectively. Only the continuous flow cores will be discussed here. (See Figure 5 for a photograph of the JCF-Z rotor. Its continuous flow cores are shown in Figure 6.) The CF-32 Ti rotor has one core (Figure 7) and is designed for continuous flow operation only. It has a maximum speed of 32,000 rpm and can be used in most Model L series ultracentrifuges, and the Optima X series ultracentrifuges.

The bowls and lids of both the JCF-Z and CF-32 Ti rotors are made of titanium. The cores are Noryl which can be used safely with most of the common density gradient media and buffers. (Consult the rotor or centrifuge instruction manuals for a list of materials to which Noryl is resistant.) All but the JCF-Z Small Pellet Core are tapered and have 4 removable Noryl baffles attached that stabilize the liquid within the rotors during operation. These baffles divide the rotor interior. (Unlike the other cores, the Small Pellet Core has 6 cavities within the core itself in which pelleting takes place.)

The amount of space between the core and the bowl wall determines the total capacity of the rotor. Of this total capacity, a certain amount can be occupied by pellet layer, gradient, or cushion at the periphery of the rotor. The remaining capacity, next to the core, is called the taper volume. This is the region through which the starting material flows, and where sedimentation into a gradient or onto the cushion or rotor wall takes place.

The specifications of the various rotor-core configurations are summarized in Table 1.

JCF-Z Rotor
The JCF-Z rotor equipped with one of its continuous flow cores is suitable for: the sedimentation of bacteria; the larger subcellular particles such as membranes, mitochondria, etc.; and those viruses or other particles that have sedimentation coefficients of 500 S and higher. It is also used for clearing virus-containing culture media of cellular debris prior to final collection of the virus, by pelleting or banding in the same rotor or the CF-32 Ti. When running at its maximum speed of 20,000 rpm, the JCF-Z with the Standard Core generates 32,000 x g at the core bottom and 39,900 x g at the inner surface of the bowl wall. It can be operated at flow rates between 3 and 45 L/h (the rotating seal assembly requires a minimum flow rate of 3 L/h). A high-flow seal assembly is available for handling very large volumes where higher flow rates would be expedient. This accessory makes it possible to process up to 100 L/h.

This rotor can be used in select Avanti centrifuges without modification. These centrifuges are refrigerated, but precooling the rotor is recommended whenever temperature control of the sample is critical. In this case, the fluids to be pumped through the rotor should be precooled also because they will be warmed to some extent as they travel through the pump tubing and the seal assembly. Sample reservoirs should be kept in ice or in a refrigerated water bath, and tubing lines should be kept as short as possible. The amount of the anticipated pellet, or the necessity to sediment onto a cushion or into a step gradient, determines which of the three JCF-Z continuous flow cores should be used.

Figure 6
Figure 6: JCF-Z continuous flow rotor and cores..
Figure 7
Figure 7: CF-32 Ti continuous flow rotor and core.
The Standard Core has a total capacity of 660 mL. Of this volume, about 400 mL can be occupied by pellet, step gradient, or cushion. This core is suitable for pelleting against a cushion or the rotor wall, clarifying liquids, or banding of particles.

 

The Large Pellet Core is suitable for pelleting only. It has a total capacity of 1250 mL, of which approximately 800 mL can be occupied by pellet. This core is well-suited for centrifuging fluids that contain a large amount of solids— a solid-to-liquid ratio as high as 1:2 can be processed. Of course, a high solid-to-liquid ratio means that less material can be processed before stopping to unload the rotor.

The Small Pellet Core has a total capacity of 240 mL, of which about 200 mL can be occupied by pellet. It is designed for processing large amounts of material with a low solid-to-liquid ratio (water that contains algae or clay particles, for example). In order to minimize resuspension of sedimented material, this core has 6 individual cavities, each holding removable canoe-shaped containers in which the pellets are collected. This core, too, is designed for pelleting only.

The JCF-Z rotor has two other interchangeable cores for zonal separations: the Reorienting Gradient Core and the Zonal Core.

CF-32 Ti Rotor
The CF-32 Ti rotor can be used to concentrate particles with a wide range of sizes. Because of its high speed (32,000 rpm), it is particularly useful for banding viruses or other small particles that have sedimentation coefficients as small as 50 S. At maximum speed, it generates 86,100 x g and 102,000 x g at the core bottom and inner surface of the bowl wall, respectively. It operates at flow rates up to 9 L/h.

The total capacity of this rotor is 430 mL, of which about 330 mL can be occupied by pellet. This capacity is reduced when a cushion or step gradient is used, either of which will occupy about 300 mL. The ultracentrifuges that accommodate the CF-32 Ti rotor provide refrigerated operation. Because the seal assembly of the CF-32 Ti is equipped with a stainless steel water jacket, normal temperatures at the seal can be maintained at much lower flow rates than in the JCF-Z rotor. A tap water supply can be used for the water jacket unless a temperature of 10ºC or lower at the seal assembly is necessary. In that case, a water cooler and pump should be used, and the starting material and rotor precooled as well.

Specifications CF-32-Ti JCF-Z
  Standard Core Small Pellet Core Large Pellet Core
Maximum Rotor Speed 32,000 rpm1 20,000 rpm 20,000 rpm 20,000 rpm
Maximum Force at Bottom of Core 86,100 g 32,000 g 25,000 g 23,000 g
Maximum Force at Top of Core 91,950 g 34,000 g 25,000 g 25,000 g
Maximum Force at Rotor Wall 102,000 g 9,900 g 6,300 g 39,900 g
Total Rotor Capacity 430 mL 660 mL 240 mL 1250 mL
Maximum Pellet Capacity 330 mL 400 mL 204 mL 800 mL
Maximum Flow Rate—Standard Seal Assembly 9 L/h 45 L/h 45 L/h 45 L/h
Maximum Flow Rate—High Flow Seal Assembly 100 L/h 100 L/h 100 L/h
Maximum Permissible Density of Contents at Maximum Speed2 1.20 g/mL 1.45 g/mL 1.45 g/mL 1.45 g/mL
Permissible pH Range for Liquids pH 4–10 pH 4–10 pH 4–10 pH 4–10

1 After 1000 runs or 2500 hours of centrifugation, the CF-32 Ti must be derated to 29,000 rpm.
2 If the density of the heaviest gradient fraction exceeds 1.20 g/mL for the CF-32 Ti, or 1.45 g/mL for the JCF-Z, the maximum permissible rotor speed must be reduced. Speed reductions can be calculated as follows:

Equation 8

Equation 9
Speed reductions may also be required when sedimenting unusually heavy pellets, i.e., liquids containing metal or clay particles, for example.

Other Equipment Required

In addition to either the JCF-Z or CF-32 Ti rotor and its respective centrifuge, a peristaltic pump—capable of operating against a back pressure of 138 kPa (20 psi)—is required for introducing cushions or step gradients into the rotor, as well as for pumping starting material. A pressure gauge to check flow rates between the pump and the seal assembly may also be helpful. A flow-through photometer is useful for monitoring the effluent to observe sample cleanout during pelleting, and for identifying banded material during unloading of cushions or gradients. Tubing lines in and out of the flow cell should have the same diameter as the rotor tubing. A fraction collector may also be desirable. Of course, it will be necessary to have sample and effluent reservoirs, as well as any refrigeration accessories required. Typical equipment setups are shown in Figures 8 and 9.

Figure 8
Figure 8: Equipment arrangement for loading and unloading cushion or step gradient.
Figure 9
Figure 9: Equipment arrangement during continuous flow centrifugation.

 

Typical Questions and Answers Regarding Continuous Flow Centrifugation

Q: I frequently need to separate particles from large volumes of starting material, but I’m not sure if I need a continuous flow rotor or a zonal rotor. Which would be better for my application?
A: If you have been successfully using conventional fixed-angle or swinging bucket rotors to pellet your particles, with or without a cushion, a continuous flow rotor will enable you to scale-up this process. This will also be true if you have been banding the par ticles isopycnically, provided there are no contaminants which band near the same density. If you do need to resolve multicomponent samples, one of the zonal rotors would be a better choice. Although the sample handling capabilities of zonal rotors (20–200 mL) are considerably less than continuous flow rotors, they are 50–100 times greater than those of swinging bucket rotors.

Also, if you have been isolating your particles by ratezonal sedimentation, or need to obtain sedimentation coefficients for your particles, a zonal rotor is what you need. Continuous flow rotors are not suitable for rate zonal centrifugation.

 
Q: I am currently using a fixed-angle rotor for pelleting and would like to make the same separation by continuous flow methods. How do I determine the proper flow rate?
A: In order to determine the necessary flow rate, you must first calculate an approximate sedimentation coefficient for the par ticle of interest. For this purpose, a rough estimate is sufficient and can be calculated as follows:

s = k/t

where s is the sedimentation coefficient in Svedberg units, k is the k-factor (clearing factor) for the fixedangle rotor you are currently using (it can be found in your rotor manual), and t is the time (in hours) it takes to pellet the particles in that rotor. If your rotor was operated at less than maximum speed, determine s as follows:

s = (k/t) (maximum rotor speed/reduced rotor speed)2

The estimated sedimentation coefficient can now be used to obtain an approximate flow rate from the nomograms shown on pages 11 and 12.

 
Q: How serious are air bubbles?
A: Air bubbles will cause high back-pressure. This is not a problem at low speed, as during loading; but at operating speed, the buoyancy of the bubble is multiplied by the relative centrifugal force. Such a bubble cannot be dislodged by inflowing sample. If the pressure is above 173 kPa (25 psig), follow the instructions given in the rotor manual for removing air trapped in the system. It may be necessary to install a bubble trap between the pump and the rotor connection to collect any air bubbles which may have formed in the sample due to outgassing.
 
Q: How do I determine run conditions?
A: Run conditions are a combination of speed and flow rate. Unless a sample particle is so small that it requires the highest possible speed with the lowest practical flow rate, there can be multiple speed/flow rate combinations that will retain particles of a given size class. Usually flow rate is chosen based on the amount of sample to be processed and how long you want that processing to take—speed then follows from that choice.
 
Q: How long does it take to set up for a continuous flow separation?
A: An experienced user will need about 30 minutes to prepare the equipment and about 10 minutes to dismantle it.
 
Q: How do I get my sample out?
A: Some samples, i.e., bacteria or yeast, will pellet on the rotor wall. Remove the supernatant remaining with a large syringe or siphon and scrape out the pellet cake. If you are using the JCF-Z Small Pellet Core, simply remove the canoe-shaped containers and scrape out the pellet. Other samples, such as cell membranes or whole cells, will not adhere to the rotor wall but will form a very concentrated slurry with the liquid remaining in the bowl. In this case, an additional spin in a conventional rotor will yield a pellet.
 
Q: How should I clean my rotor after use?
A: After each use, all parts of the rotor and seal assembly that have come in contact with the sample should be rinsed in mild detergent, followed by distilled water. They should be dried with warm air. They may then be autoclaved at temperatures up to 125ºC, except for the removable Noryl baffles on the core and the canoe-shaped containers of the Small Pellet Core. These may warp at this temperature. Moisture can reduce the lifetime of the JCF-Z bearings, so they should be placed in a vapor-tight container if autoclaved.
 
Q: What about continuous flow centrifugation of pathogens?
A: All Beckman Coulter continuous flow rotor manuals carry this warning: “The processing of large volumes of pathogenic materials in these rotors can produce extremely potent concentrations. Agents that are relatively harmless in their naturally occurring dilute state may become infectious when concentrated. Biological hazards must be carefully assessed and precautions taken.” Remote control panels are available for many of the centrifuges, and Absolute High Efficiency Particulate Aerosol (HEPA) filters are also available for placing between the vacuum chamber and the vacuum pump. Consideration must be given to the airflow in the room, and your Laboratory Safety Officer should be consulted.

 

If high-risk agents are being investigated, the use of a Class III biological safety cabinet with a modified centrifuge should be considered. Beckman Coulter will be pleased to work with you on the design of an appropriate system.

 
Q: What should I do if I am unsure of the condition of my continuous flow rotor?
A: Beckman Coulter, Inc., will provide free inspection of any rotor at the request of the user. Rotor owners are urged to make use of this service if they have any doubts about the condition of a rotor.

Figure 10
JCF-Z Rotor Nomogram. Theoretical maximum flow rate for 100% cleanout when using Standard Core. To use, place a ruler on the page to intersect the middle column (known Svedberg units). Pivot the ruler about this point to intersect the other two columns. The nomogram covers all practical combinations of speed and flow rate.
Figure 11
CF-32 Ti Rotor Nomogram. Theoretical maximum flow rate for 100% cleanout. To use, place a ruler on the page to intersect the right-hand column (known Svedberg units). Pivot the ruler about this point to intersect the other two columns. The nomogram covers all practical combinations of speed and flow rate.

 

Bibliography of Applications

References to some typical uses of the CF-32 Ti and JCF-Z continuous flow rotors are given below. (Some of the earlier literature mentions the CF-35 Ti rotor. This rotor is now designated the CF-32 Ti, and operates at a maximum speed of 32,000 rpm.) The references are grouped according to the material separated; the rotor used and the type of separation are given for each one.

Algae
Chlamydomonas reinhardtii JCF-Z, pelleted.
Schleicher M, Lukas TJ, Watterson DM. Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii. Arch. Biochem. Biophys. 229; 33–42: (1984).

Synechococcus cedrorum JCF-Z, pelleted.
Newman PJ, Sherman LA. Isolation and characterization of photosystem I and II membrane particles from the blue-green algae, Synechococcus cedrorum. Biochim. Biophys. Acta. 503; 343–361: (1978).

Bacteria
Acholeplasma laidlawii JCF-Z, pelleted.
Eriksson P-O, Rilfors L, Wieslander A, Lundberg A, Lindblom G. Order and dynamics in mixtures of membrane glucolipids from Acholeplasma laidlawii studied by 2H NMR. Biochemistry. 30; 4916–4924: (1991).

Bacillus licheniformis JCF-Z, pelleted.
Opheim D, Bernlohr RW. Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis. J. Bacteriol. 116; 1150–1159: (1973).

Opheim D, Bernlohr RW. Purification and regulation of fructose-1, 6 bisphosphatase from Bacillus licheniformis. J. Biol. Chem. 250; 3024–3033: (1975).

Opheim DJ, Bernlohr RW. Fructose-1,6-bisphosphatase from Bacillus licheniformis. Methods in Enzymology. Vol. 90; pp. 384–391. Edited by WA Wood. New York, Academic Press, 1982.

Coxiella burneti antigens JCF-Z, banded in sucrose.
Leyk W, Krauss H. Zur Reinigung von Coxiella burneti- Antigen mittels Dichtegradienten-zentrifugation. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg., Abt. 1: Orig., Reihe A. 230; 508–517: (1975).

Escherichia coli JCF-Z, pelleted.
Meyers M, Blasi F, Bruni CB, Deeley RG, Kovach JS, Levinthal M, Mullinix KP, Vogel T, Goldberger RF. Specific binding of the first enzyme for histidine biosynthesis to the DNA of the histidine operon. Nucleic Acids Res. 2; 2021–2036: (1975).

Scherzer E, Auer B, Schweiger M. Identification, purification, and characterization of Escherichia coli virus T1 DNA methyl-transferase. J. Biol. Chem. 262; 15225–15231: (1987).

Wagenknecht T, Bloomfield VA. In vitro polymerization of bacteriophage T4D tail core subunits. J. Mol. Biol. 116; 347–359: (1977).

Zidwick MJ, Keller G, Rogers P. Regulation and coupling of argECBH mRNA and enzyme synthesis in cell extracts of Escherichia coli. J. Bacteriol. 159; 640–646: (1984).

Salmonella typhimurium JCF-Z, pelleted.
Robertson DE, Kroon PA, Ho C. Nuclear magnetic resonance and fluorescence studies of substrateinduced conformational changes of histidine-binding protein J of Salmonella typhimurium. Biochemistry. 16; 1443–1451; (1977).

Spirochetes JCF-Z, pelleted.
Livermore BP, Johnson RC. Lipids of the Spirochaetales: comparison of the lipids of several members of the genera Spirochaeta, Treponema, and Leptospira. J. Bacteriol. 120; 1268–1273: (1974).

Staphylococcus aureus JCF-Z, pelleted.
Peterson PK, Wilkinson BJ, Kim Y, Schmeling D, Douglas SD, Quie PG, Verhoef J. The key role of peptidoglycan in the opsonization of Staphylococcus aureus. J. Clin. Invest. 61; 597–609: (1978).

Streptococcus pyogenes JCF-Z, culture fluid clarified.
Cunningham CM, Barsumian EL, Watson DW. Further purification of group A streptococcal pyrogenic exotoxin and characterization of the purified toxin. Infect. Immun. 14; 767–775: (1976).

Other Cells
Acanthamoeba castellanii JCF-Z, pelleted.
Radebaugh CA, Matthews JL, Geiss GK, Liu F, Wong J-M, Bateman E, Camier S, Sentenac A, Paule MR. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-1B (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors. Mol. Cell. Biol. 14; 597–605: (1994).

Crithidia luculiae JCF-Z, pelleted.
Steenkamp DJ. The purine-2-deoxyribonucleosidase from Crithidia luculiae. Purification and trans-N-deoxyribosylase activity. Eur. J. Biochem. 197; 431–439: (1991).

Virus-infected Vero cells JCF-Z, pelleted.
Barrett N, Mitterer A, Mundt W, Eibl J, Eibl M, Gallo RC, Moss B, Dorner F. Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity. AIDS Res. Hum. Retroviruses. 5; 159–173: (1989).

Cell Culture Media
Calf serum CF-32 Ti, clarified.
Uckert W, Wunderlich V, Bender E, Sydow G, Bierwolf D. The protein pattern of PMF virus, a type-D retrovirus from malignant permanent human cell lines. Arch. Virol. 64; 155–166: (1980).

Cell culture medium CF-32 Ti, clarified.
Henderson LE, Hewetson JF, Hopkins RF III, Sowder RC, Neubauer RH, Rabin H. A rapid, large scale purification procedure for gibbon interleukin 2. J. Immunol. 131; 810–815: (1983).

Iwata KK, Fryling CM, Knott WB, Todaro GJ. Isolation of tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line. Cancer Res. 45; 2689– 2694: (1985).

Marquardt H, Todaro GJ. Human transforming growth factor. Production by a melanoma cell line, purification, and initial characterization. J. Biol. Chem. 257; 5220– 5225: (1982).

Marquardt H, Wilson GL, Todaro GJ. Isolation and characterization of a multiplication-stimulating activity (MSA)-like polypeptide produced by a human fibrosarcoma cell line. J. Biol. Chem. 255; 9177–9181: (1980).

Cell culture medium JCF-Z, clarified.
Marquardt H, Wilson GL, Todaro GJ. Isolation and characterization of a multiplication-stimulating activity (MSA)-like polypeptide produced by a human fibrosarcoma cell line. J. Biol. Chem. 255; 9177–9181: (1980).

Miscellaneous
Brain protein and protein precipitates JCF-Z, clarified, pelleted.
Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 35; 349–358: (1983).

Brain protein precipitates JCF-Z, pelleted.
Turk E, Teplow DB, Hood LE, Prusiner SB. Purification and properties of the cellular and scrapie hamster prion proteins. Eur. J. Biochem. 176; 21–30: (1988).

Scrapie-containing precipitates JCF-Z, pelleted.
Prusiner SB, McKinley MP, Bolton DC, Bowman KA, Groth DF, Cochran SP, Hennessey EM, Braunfeld MB, Baringer JR, Chatigny MA. Prions: methods for assay, purification, and characterization. Methods in Virology. Vol. 8; pp. 293–345. Edited by K Maramorosch and H Koprowski. Orlando, Academic Press, 1984.

Surface water JCF-Z, cleared of particulates.
Reid PM, Wilkinson AE, Tipping E, Jones MN. Determination of molecular weights of humic substances by analytical (UV scanning) ultracentrifugation. Geochim. Cosmochim. Acta. 54; 131–138: (1990).

Subcellular Fractions
Brain homogenate JCF-Z, clarified.
Prusiner SB, McKinley MP, Bolton DC, Bowman KA, Groth DF, Cochran SP, Hennessey EM, Braunfeld MB, Baringer JR, Chatigny MA. Prions: methods for assay, purification, and characterization. Methods in Virology. Vol. 8; pp. 293–345. Edited by K Maramorosch and H Koprowski. Orlando, Academic Press, 1984.

Turk E, Teplow DB, Hood LE, Prusiner SB. Purification and properties of the cellular and scrapie hamster prion proteins. Eur. J. Biochem. 176; 21–30: (1988).

Gap Junctions, Rat liver JCF-Z, pelleted.
Hertzberg EL, Gilula NB. Isolation and characterization of gap junctions from rat liver. J. Biol. Chem. 254; 2138– 2147: (1979).

Lysate, Human platelet JCF-Z, supernatant clarified.
Heldin C-H, Johnsson A, Ek B, Wennergren S, Rönnstrand L, Hammacher A, Faulders B, Wasteson A, Westermark B. Purification of human platelet-derived growth factor. Methods in Enzymology. Vol. 147; pp. 3–13. Edited by D Barnes. Orlando, Academic Press, 1987.

Membranes, Human erythrocyte JCF-Z, banded on sucrose cushion.
Kahane I, Furthmayr H, Marchesi VT. Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents. Biochim. Biophys. Acta. 426; 464–476; (1976).

Marchesi VT. Isolation of spectrin from erythrocyte membranes. Methods in Enzymology. Vol. 32; pp. 275– 277. Edited by S Fleischer and L Packer. New York, Academic Press, 1974.

Mitochondria, Beef liver JCF-Z, pelleted.
Laipis PJ, Hauswirth WW, O’Brien TW, Michaels GS. A physical map of bovine mitochondrial DNA from a single animal. Biochim. Biophys. Acta. 565; 22–32: (1979).

Mitochondria, Fungus JCF-Z, pelleted.
Brambl R. Mitochondrial biogenesis during fungal spore germination. Biosynthesis and assembly of cytochrome c oxidase in Botryodiplodia theobromae. J. Biol. Chem. 255; 7673–7680: (1980).

Josephson M, Brambl R. Mitochondrial biogenesis during fungal spore germination. Purification, properties and biosynthesis of cytochrome c oxidase from Botryodiplodia theobromae. Biochim. Biophys. Acta. 606; 125–137: (1980).

Nuclei, Calf liver CF-32 Ti, pelleted through step sucrose gradient.
Cacace MG, Nucci R, Reckert H. Large scale preparation of calf liver nuclei by continuous flow centrifugation. Experientia. 33; 855–857: (1977).

Plasma membranes, Beef liver JCF-Z, pelleted.
Rosen P, Ehrich B, Junger E, Bubenzer HJ, Kühn L. Binding and degradation of insulin by plasma membranes from bovine liver isolated by a large scale preparation. Biochim. Biophys. Acta. 587; 593–605: (1979).

Plasma membranes, Pig liver JCF-Z, pelleted.
Meyer HE, Bubenzer H-J, Herbertz L, Kuehn L, Reinauer H. Purification of the insulin receptor protein from porcine liver membranes. Hoppe-Seyler’s Z. Physiol. Chem. 362; 1621–1629: (1981).

Plasma membranes, Rat liver JCF-Z, pelleted.
Hertzberg EL. Isolation and characterization of liver gap junctions. Methods in Enzymology. Vol. 98; pp. 501– 510. Edited by S Fleischer and B Fleischer. New York, Academic Press, 1983.

Tissue filtrate, Mouse liver JCF-Z, pelleted.
Goodenough DA. Bulk isolation of mouse hepatocyte gap junctions. Characterization of the principal protein, connexin. J. Cell Biol. 61; 557–563: (1974).

Viruses
Calf diarrhea CF-32 Ti, banded in sucrose.
Sharpee RI, Mebus CA, Bass EP. Characterization of a calf diarrheal coronavirus. Am. J. Vet. Res. 37; 1031– 1041: (1976).

Epstein-Barr JCF-Z, virus-containing culture fluid clarified; CF-32 Ti, pelleted.
Shibley GP, Manousos M, Munch K, Zelljadt I, Fisher L, Mayyasi S, Harewood K, Stevens R, Jensen KE. New method for large-scale growth and concentration of the Epstein-Barr viruses. Appl. Environ. Microbiol. 40; 1044–1048: (1980).

Feline leukemia JCF-Z, virus-containing culture fluid clarified; CF-32 Ti, banded in sucrose.
Hoekstra J, Deinhardt F. Simian sarcoma and feline leukemia virus antigens: isolation of species- and interspeciesspecific proteins. Intervirology. 2; 222–230: (1973–74).

Salerno RA, Larson VM, Phelps AH, Hilleman MR. Infection and immunization of cats with the Kawakami-Theilen strain of feline leukemia virus. Proc. Soc. Exp. Biol. Med. 160; 18–23: (1979).

Gibbon ape lymphoma CF-32 Ti, banded in sucrose.
Harewood KR, Chang P, Higdon C, Larson D. The endogenous reverse transcriptase activity of gibbon ape lymphoma virus: characterization of the DNA product. Biochim. Biophys. Acta. 407; 14–23: (1975).

Harvey murine sarcoma JCF-Z, virus-containing culture fluid clarified; CF-32 Ti, banded in sucrose.
Parks WP, Scolnick EM. In vitro translation of Harvey murine sarcoma virus RNA. J. Virol. 22; 711–719: (1977).

Hepatitis A CF-32 Ti, pelleted.
Wheeler CM, Robertson BH, Van Nest G, Dina D, Bradley DW, Fields HA. Structure of the hepatitis A virion: peptide mapping of the capsid region. J. Virol. 58; 307–313: (1986).

Hepatitis B CF-32 Ti, banded in sucrose.
Takahashi T, Nakagawa S, Hashimoto T, Takahashi K, Imai M, Miyakawa Y, Mayumi M. Large-scale isolation of Dane particles from plasma containing hepatitis B antigen and demonstration of a circular double-stranded DNA molecule extruding directly from their cores. J. Immunol. 177; 1392–1397: (1976).

Tsuda F, Takahashi T, Takahashi K, Miyakawa Y, Mayumi M. Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination. J. Immunol. 115; 834–838: (1975).

Influenza CF-32 Ti, banded in sucrose.
McAleer WJ, Hurni W, Wasmuth E, Hilleman MR. High resolution flow-zonal centrifuge system. Biotechnol. Bioeng. 21; 317–321: (1979).

Mistretta AP, Crovari-Cuneo P, Giacometti G, Sacchi G, Strozzi F. Purification and concentration of influenza inactivated viruses by continuous-flow zonal centrifugation. Boll. Ist. Sieroter. Milan. 54; 45–56: (1975).

Mountford CE, Grossman G, Hampson AW, Holmes KT. Influenza virus: an NMR study of mechanisms involved in infection. Biochim. Biophys. Acta. 720; 65–74: (1982).

Mammalian C-type CF-32 Ti, banded in sucrose.
Olpin J, Oroszlan S, Gilden RV. Biophysical-immunological assay for ribonucleic acid type C viruses. Appl Microbiol. 28; 100–105: (1974).

Oroszlan S, Bova D, Huebner RJ, Gilden RV. Major groupspecific protein of rat type C viruses. J. Virol. 10; 746– 750: (1972).

Oroszlan S, Summers MR, Foreman C, Gilden RV. Murine type-C virus group-specific antigens: interstrain immunochemical, biophysical, and amino acid sequence differences. J. Virol. 14; 1559–1574: (1974).

Moloney murine sarcoma CF-35 Ti, sedimente donto sucrose cushion.
Gilden RV, Oroszlan S, Huebner RJ. Antigenic differentiation of M-MSV(O) from mouse, hamster, and cat C-type viruses. Virology. 43; 722–724: (1971).

Mouse mammary tumor CF-32 Ti, banded in sucrose.
Drohan W, Kettmann R, Colcher D, Schlom J. Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RIII mouse strains. J. Virol. 21; 986–995: (1977).

Mouse oncorna CF-32 Ti, banded in sucrose.
Burnette WN, Riggin CH, Mitchell WM. Physical and chemical properties of an oncornavirus associated with a murine adrenal carcinoma cell line. J. Virol. 14; 110–115: (1974).

Murine leukemia CF-32 Ti, banded in sucrose; JCF-Z, virus-containing culture fluid clarified.
Sherr CJ, Todaro GJ. Purification and assay of murine leukemia viruses. Methods in Enzymology. Vol. 58; pp. 412–424. Edited by WB Jakoby and IH Pastan. New York, Academic Press, 1979.

Murine leukemia CF-32 Ti, banded in sucrose.
Chattopadhyay SK, Hartley JW, Lander MR, Kramer BS, Rowe WP. Biochemical characterization of the amphotropic group of murine leukemia viruses. J. Virol. 26; 29–39: (1978).

Chattopadhyay SK, Jay G, Lander MR, Levine AS. Correlation of the induction of transcription of the AKR mouse genome by 5-iododeoxyuridine with the activation of an endogenous murine leukemia virus. Cancer Res. 39; 1539–1546: (1979).

Johnson PA, Rosner MR. Characterization of murinespecific leukemia virus receptor from L cells. J. Virol. 58; 900–908: (1986).

Levy RL, Lerner RA, Dixon FJ. Enhancement of infectivity and oncogenicity of a murine leukemia virus in adult mice by X-irradiation. Cancer Res. 36; 2090–2095: (1976).

Zwerner RK, Wise KS, Action RT. Harvesting the products of cell growth. Methods in Enzymology. Vol. 58; pp. 221–229. Edited by WB Jakoby and IH Pastan. New York, Academic Press, 1979.

Rauscher murine leukemia JCF-Z, virus-containing culture fluid clarified.
Johnson RW, Perry A, Robinson OR Jr, Shibley GP. Method for reproducible large-volume production and purification of Rauscher murine leukemia virus. Appl. Environ. Microbiol. 31; 182–188: (1976).

RD114 CF-32 Ti, banded in sucrose.
Okabe H, Gilden RV, Hatanaka M. RD114 virus-specific sequences in feline cellular RNA: detection and characterization. J. Virol. 12; 984–994: (1973).

Retrovirus CF-32 Ti, banded in sucrose.
Faff O, Murray BA, Erfle V, Hehlmann R. Large scale production and purification of human retrovirus-like particles related to the mouse mammary tumor virus. FEMS Microbiol. Lett. 109; 289–296: (1993).

Uckert W, Sydow G, Hertline I, Rudolph M, Wunderlich V. Comparison of different methods for large volume concentration of a type D retrovirus (PMFV). Arch. Geschwulstforsch. 52; 541–549: (1982).

Retrovirus CF-32 Ti, pelleted.
Anderson KP, Low ML, Lie YS, Keller G-A, Dinowitz M. Endogenous origin of defective retroviruslike particles from a recombinant Chinese hamster ovary cell line. Virology. 181; 305–311: (1991).

Simian sarcoma type 1 and its associated virus JCF-Z, tissue culture fluid clarified; CF-32 Ti, CF-35 Ti, banded in sucrose.
Hoekstra J, Deinhardt F. Simian sarcoma and feline leukemia virus antigens: isolation of species- and interspeciesspecific proteins. Intervirology. 2; 222–230: (1973–74).

Jensik S, Hoekstra J, Silver S, Northrop RL, Deinhardt F. The 60 to 70S RNA and reverse transcriptase of simian sarcoma and simian sarcoma-associated viruses. Intervirology. 1; 229–241: (1973).

Sindbis CF-32 Ti, banded in sucrose.
von Bonsdorff C-H, Harrison SC. Hexagonal glycoprotein arrays from Sindbis virus membranes. J. Virol. 28; 578– 583: (1978).

Vaccinia JCF-Z, banded in sucrose.
Schwenen M, Richter KH. Isolierung von Vaccinia-Viren aus Tierhaut-Impfstoff. Eine vergleichende Untersuchung moderner Präparationsmethoden für die Impfstoffproduktion. II. Zonen-Zentrifugation im Sucrose-Dichtegradienten und Differentialzentrifugation. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg., Abt. 1: Orig., Reihe A. 228; 415–442: (1974).

Yeast
Saccharomyces carlsbergensis JCF-Z, pelleted.
El-Baradi TTAL, Raue HA, De Regt VCHF, Planta RJ. Stepwise dissociation of yeast 60S ribosomal subunits by LiCl and identification of L25 as a primary 26S rRNA binding protein. Eur. J. Biochem. 144; 393–400: (1984).

Saccaromyces cerevisiae JCF-Z, pelleted.
Kane PM, Yamashiro CT, Stevens TH. Biochemical characterization of the yeast vacuolar H+-ATPase. J.Biol. Chem. 264; 19236–19244: (1989).

 

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