Abstract:
A system for separating a sample and collecting the separated sample. The system includes an ultracentrifuge having a cylindrical rotor. The system includes a gradient delivery assembly for delivering a gradient solution to the ultracentrifuge rotor, and a sample delivery assembly for delivering the solution containing the sample to the ultracentrifuge rotor. he system includes a fraction collection assembly for collecting discrete volumes of the separated sample. The system includes a processor for controlling operation of the ultracentrifuge, as well as the gradient delivery assembly, the sample delivery assembly, and/or the fraction collection assembly.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     The present invention relates generally to a fraction collection system, and more particularly to an automated system permitting separation, enrichment and fractionation of sub-cellular particles.  
         [0002]     Characterization of biological materials, such as biological molecules and organelles, has become increasingly important. Precise characterization of these materials can lead to novel drug therapies for treating disease, as well as to a greater understanding of the mechanisms underlying many diseases. Many biological materials exhibit a buoyant density that can be used to distinguish them from other materials. Such materials can be separated using density gradients and procedures such as differential centrifugation. For example, lipoproteins are composed of varying amounts of proteins and lipids. They differ not only by size and electrophoretic mobility, but also by buoyant density. Thus, in addition to other techniques available for separating, identifying, and classifying lipoproteins, density-gradient ultracentrifugation may be used. Such methodologies are complicated and time consuming particularly when minute fractions of large samples are being analyzed. Thus, there is a need for an automated method of isolating biological materials that can be used with smaller or larger samples and a method that is capable of segregating dilute materials from large samples.  
       SUMMARY OF THE INVENTION  
       [0003]     Briefly, the present invention includes a system for separating a sample and collecting the separated sample. The system comprises an ultracentrifuge having a cylindrical rotor sized and shaped for holding a solution containing the sample and spinning the solution to separate the sample contained in the rotor according to its buoyant density. The system also comprises a gradient delivery assembly connected to the ultracentrifuge for delivering a gradient solution to the ultracentrifuge rotor, and a sample delivery assembly connected to the ultracentrifuge for delivering the solution containing the sample to the ultracentrifuge rotor. In addition, the system includes a fraction collection assembly connected to the ultracentrifuge for collecting discrete volumes of the separated sample. Further, the system comprises a processor for controlling operation of the ultracentrifuge and at least one assembly of a group of assemblies consisting of the gradient delivery assembly, the sample delivery assembly, and the fraction collection assembly.  
         [0004]     In another aspect, the invention includes a fraction volume measurement assembly for measuring and dispensing a predetermined volume of a sample. The assembly comprises a measurement tube having a lower end and an upper end. The lower end includes an opening for permitting the sample to enter and leave the tube. The assembly also comprises a valve in communication with the opening in the lower end of the measurement tube for controlling flow of the sample into and out of the tube. Further, the assembly comprises an electromagnetic transmitter mounted on the tube for transmitting electromagnetic energy along a line, and an electromagnetic receiver mounted on the tube opposite the transmitter and along the line of transmitted electromagnetic energy so that when an upper level of the sample in the tube is below the line, energy transmitted by the transmitter is received by the receiver and when an upper level of the sample in the tube is above the line, the transmitted energy is retracted so it is not received by the receiver, indicating the predetermined volume of the sample is present in the tube.  
         [0005]     In yet another aspect, the invention includes a fraction collection assembly for collecting fractions having predetermined volumes. The assembly comprises a measurement tube having a lower end and an upper end. The lower end includes an opening for permitting the sample to enter and leave the tube. The assembly also comprises a valve in communication with the opening in the lower end of the measurement tube for controlling flow of the sample into and out of the tube, and an infrared sensor mounted adjacent the tube for determining when a predetermined volume of sample is present in the tube.  
         [0006]     In still another aspect, the invention includes a processor for automatically controlling operation of a system including an ultracentrifuge having a rotor for containing and spinning a solution to separate particles in the solution according to their buoyant density, a solution delivery assembly connected to the ultracentrifuge for delivering a solution to the ultracentrifuge rotor, and a sample delivery assembly connected to the ultracentrifuge for delivering the solution containing the particles to the ultracentrifuge rotor. The processor provides commands to the system to perform a method comprising filling the rotor of the ultracentrifuge with buffer solution, and eliminating air from the rotor of the ultracentrifuge. The method also comprises filling at least a portion of the rotor of the ultracentrifuge with gradient solution, rotating the rotor to a predetermined speed, and loading a sample solution into the rotating rotor. In addition, the method comprises recycling eluent through the rotating rotor, stopping the rotor, drawing solution from a lower end of the stopped rotor, and indexing a receptacle having a series of wells to sequentially collect the solution from the lower end of the rotor in the wells.  
         [0007]     Other features of the present invention will be in part apparent and in part pointed out hereinafter. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]      FIG. 1  is a schematic diagram of a system of the present invention;  
         [0009]      FIG. 2  is a schematic diagram of a fraction volume measurement assembly of the present invention;  
         [0010]      FIG. 3  is a perspective of a fraction volume measurement assembly;  
         [0011]      FIG. 4  is a perspective of the fraction volume measurement assembly shown in  FIG. 3  rotated and partially disassembled;  
         [0012]      FIG. 5  is a rotated perspective of the assembly shown in  FIG. 3 ; and  
         [0013]      FIG. 6  is a separated view of a portion of the fraction volume measurement assembly. 
     
    
       [0014]     Corresponding reference characters indicate corresponding parts throughout the several views of the drawings.  
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0015]     Referring now to the drawings and in particular to  FIG. 1 , a system of the present invention is designated in its entirety by the reference numeral  20 . The system  20  includes an ultracentrifuge  22  having a cylindrical rotor  24  that spins about a vertically-oriented axis at high speeds (e.g., speeds as high as 35,000 revolutions per minute (rpm) or more) to separate particles contained in solution in the rotor according to their buoyant density. The rotor  24  has a sample port  26  at its lower end through which samples are introduced and withdrawn. An overflow port  28  is provided at an upper end of the rotor  24 . An electric motor (not shown) controlled by a processor  30  is used to spin the rotor  24 . Although other processors may be used without departing from the scope of the present invention, in one embodiment the processor is a CFP-2020 compact field point network module available from National Instruments Corporation of Austin, Tex. As will be appreciated by those skilled in the art, the processor  30  may include a main processor and one or more input-output processors or modules. The ultracentrifuge  22  may also include a cooling system (not shown) to maintain components of the ultracentrifuge within operating limits. Although other ultracentrifuges  22  may be used without departing from the scope of the present invention, in one embodiment the ultracentrifuge is a pCFU ultracentrifuge available from Alfa Wassermann, Inc. of West Caldwell, N.J. Because other features of the ultracentrifuge  22  are well known to those skilled in the art, they will not be described in further detail.  
         [0016]     The system also includes a solution delivery assembly  32 , a sample delivery assembly (generally designated by  34 ), a fraction collection assembly (generally designated by  36 ) and an overflow management assembly (generally designated by  38 ). The solution delivery assembly  32  includes a manifold  40  having five ports  42 ,  44 ,  46 ,  48 ,  50  that are selectively connected by three interconnected 3-way servo-driven stopcocks or valves  52 ,  54 ,  56 . As will be appreciated by those skilled in the art, each of the stopcocks  52 ,  54 ,  56  includes a generally cylindrical body having passages forming a tee-intersection. The stopcocks  52 ,  54 ,  56  may be rotated to align the passages so they are connected to selected ports  42 ,  44 ,  46 ,  48 ,  50 . Each of the stopcocks  52 ,  54 ,  56  is driven between the various rotational positions by a corresponding servomotor that is connected to the processor  30 . Although other manifolds may be used without departing from the scope of the present invention, in one embodiment the manifold  40  is a unit containing three ganged stopcocks available from Elcam Medical Inc. of Hackensack, N.J. Each of the stopcocks is driven by a HSR-5997TG servomotor available from Hitec RCD USA, Inc. of Poway, Calif. As will be appreciated by those skilled in the art, the servomotors position may be controlled by a pulse width modulated signal sent to an embedded controller. Confirmation that the servomotor reached the predetermined position is achieved by monitoring the electrical current drawn by the servomotor. The current approaches zero as the servomotor approaches the predetermined position. Because other features of the manifold  40  are well known to those skilled in the art, they will not be described in further detail. Port  42  is connected to a gradient reservoir  62  containing an appropriate gradient solution such as a 60% sucrose solution. Port  44  is connected to a motor-driven gradient syringe  64  having a barrel and a plunger slidably mounted in the barrel for selectively drawing gradient solution into the barrel and forcing the gradient solution out of the barrel. Port  46  is connected to a buffer reservoir  66  containing an appropriate buffer solution such as an 8% sucrose solution. Port  48  is connected to a motor-driven buffer syringe  68  having a barrel and a plunger similar to those of the gradient syringe  64  for selectively drawing buffer solution into the barrel and forcing the buffer solution out of the barrel. Both plungers of the gradient syringe  64  and the buffer syringe  68  may be driven between the various positions by a corresponding stepper motor connected to the processor  30 . Although other automated syringes may be used without departing from the scope of the present invention, in one embodiment the gradient syringe  64  and the buffer syringe  68  are 009760 100 ml glass syringes available from SGE, Incorporated of Austin Tex., driven by PK246PB stepper motors available from Oriental Motor U.S.A. Corp. of Torrance, Calif. Port  50  forms an outlet port for the solution delivery assembly  32 .  
         [0017]     The sample delivery assembly  34  includes a manifold  70  similar to the manifold  40  of the solution delivery assembly  32 . The manifold  70  has five ports  72 ,  74 ,  76 ,  78 ,  80  that are selectively connected by three interconnected 3-way servo-driven stopcocks  82 ,  84 ,  86  controlled by the processor  30 . Because the manifold  70  of the sample delivery assembly  34  is similar to the manifold  40  of the solution delivery assembly  32 , it will not be described in further detail. The outlet port  50  of the solution delivery assembly  32  is connected to port  72  of the sample deliver assembly manifold  70 . Port  74  is connected to a peristaltic sample pump  94 , and port  76  is connected to a peristaltic recycle pump  96 . Both the sample pump  94  and the recycle pump  96  are controlled by the processor  30 . Although other pumps may be used without departing from the scope of the present invention, in one embodiment both the sample pump  94  and the recycle pump  96  are 040.NP10.4DO-100RPM 314 VDL/D peristaltic pumps available from Watson-Marlow Bredel Inc. of Wilmington, Mass. Because the pumps  94 ,  96  are conventional, they will not be described in further detail. Port  78  is connected to a waste reservoir  98 , and port  80  forms an outlet port for the sample delivery assembly  34 . The sample pump  94  is connected to a sample reservoir  100  containing an appropriate sample to be separated such as a protein solution. The recycle pump  96  is connected to a recycle reservoir  102 .  
         [0018]     The fraction collection assembly  36  includes a manifold  110  similar to the manifold  40  of the solution delivery assembly  32 . The manifold  110  has five ports  112 ,  114 ,  116 ,  118 ,  120  that are selectively connected by three interconnected 3-way servo-driven stopcocks  122 ,  124 ,  126  controlled by the processor  30 . Because the manifold  110  of the fraction collection assembly  36  is similar to the manifold  40  of the solution delivery assembly  32 , it will not be described in further detail. The outlet port  80  of the sample delivery assembly  34  is connected to port  112  of the fraction collection assembly manifold  110 . Port  114  is connected to a priming accumulator  134 . Port  116  is connected to a fraction volume measurement assembly, generally designated by  130 , that is connected to a fraction collector, generally designated by  132 . Although other fraction collectors may be used without departing from the scope of the present invention, in one embodiment the collector  132  is a motor driven stage which receives a collection receptacle having a plurality of wells arranged in a grid. The position of the stage is controlled by the processor  30 . Although the Because stages and receptacles of this type are known in the art, they will not be described in further detail. Port  118  is capped to provide future system expansion. Port  120  forms an outlet port of the fraction collection assembly  36  and is connected to sample port  26  of the ultracentrifuge rotor  24 .  
         [0019]     The overflow management assembly  38  includes a manifold  140  similar to the manifold  40  of the solution delivery assembly  32 . The manifold  140  has five ports  142 ,  144 ,  146 ,  148 ,  150  that are selectively connected by three interconnected 3-way stopcocks  152 ,  154 ,  156  driven by servomotors connected to the processor  30 . Because the manifold  140  of the overflow management assembly  38  is similar to the manifold  40  of the solution delivery assembly  32 , it will not be described in further detail. The overflow port  28  of the ultracentrifuge rotor  24  is connected to port  142  of the overflow management assembly manifold  140 . Port  144  is connected to a vent  164 . Port  146  is connected to the recycle reservoir  102  of the sample delivery assembly  34 . Port  148  is connected to the waste reservoir  98 , which is connected to port  78  of the sample delivery assembly manifold  70 . Port  150  is connected to a final flow reservoir  160 .  
         [0020]     As illustrated in  FIG. 2 , the fraction volume measurement assembly  130  includes a transparent central tube  170  having a lower end  172  and an upper end  174 . Although the tube  170  may have other shapes and dimensions without departing from the scope of the present invention, in one embodiment the tube  170  has an inner diameter of about ten millimeters. A 3-way servo-driven stopcock  176  is positioned at the lower end  172  of the tube  170  and a 3-way servo-driven stopcock  178  is positioned at the upper end  174  of the tube  170 . The lower stopcock  176  has ports  182 ,  184 ,  186 . Port  182  is connected to the lower end  172  of the central tube  170 . Port  184  is connected to port  120  of the fraction collection assembly manifold  110 , and port  186  drains into the fraction collector  132 . The upper stopcock  178  has ports  190 ,  192 ,  194 . Port  190  is connected to the upper end  174  of the central tube  170 . Port  192  is connected to a vent line  196  and port  194  is connected to a lower pressure pas source  198 . Although the gas source  198  may be at other pressures without departing from the scope of the present invention, in one embodiment the gas source pressure is between about 1 pounds per square inch (psi) and about 3 psi. Although the gas source  198  may include other gases without departing from the scope of the present invention, in one embodiment the gas source provides filtered clean air.  
         [0021]     A pair of infrared transmitters  200 ,  202  are provided on one side of the central tube  170 , and a pair of corresponding infrared receivers  204 ,  206  are provided on the side of the tube opposite the transmitters. When solution inside the central tube  170  rises to a level high enough that it extends above a line between transmitter  200  and receiver  204 , the infrared signal produced by the transmitter is refracted so it does not reach the receiver. The receiver  204  sends a signal to the processor  30  indicating a corresponding level in the tube  170  has been reached and the solution in the tube has reached a corresponding volume. As will be appreciated by those skilled in the art, a volume of the tube  170  below this signal level may be adjusted by moving the transmitter and receiver pair. As will further be appreciated, the second transmitter  202  and receiver  206  may be used to measure a second predetermined solution volume. Although other transmitters and receivers may be used without departing from the scope of the present invention, in one embodiment the transmitters and receivers are photomicrosensors such as an EE-SPX613 photomicrosensor available from Omron Electronics LLC of Schaumburg, Ill.  
         [0022]     As shown in  FIG. 3 , the fraction volume measurement assembly  130  comprises a base plate  210  having a recess  212  for receiving a separable tube and stopcock sub-assembly, generally designated by  214 , which includes the central tube  170  and stopcocks  176 ,  178 . Although other tube and stopcock sub-assemblies may be used without departing from the scope of the present invention, in one embodiment the sub-assemblies are specially made by Elcam Medical Inc. of Hackensack, N.J. A spring loaded clamp  216  is provided on the base plate  210  for holding the tube and stopcock sub-assembly  214  in position in the recess  212 . As illustrated in  FIG. 4 , the assembly  130  includes cups  220 ,  222  adapted to receive levers on the stopcocks  176 ,  178 , respectively, when the sub-assembly  214  is in position in the recess  212 . The cups  220 ,  222  are rotated by corresponding servomotors  224 ,  226  mounted on a bracket  228  connected to the base plate  210 .  FIG. 5  provides an alternate view of the servomotors  224 ,  226  and bracket  228 . As illustrated in  FIG. 6 , the infrared transmitters  200 ,  202  and receivers  204 ,  206  are held in a mount  230 . The mount  230  is biased by springs  232  toward the tube and stopcock sub-assembly  214  when it is positioned in the recess  212 . Further, the mount  230  is fixed to an adjustment mechanism, generally designated by  234  ( FIG. 5 ), which permits the positions of the transmitters  200 ,  202  and receivers  204 ,  206  to be adjusted relative to the tube and stopcock sub-assembly  214 , permitting adjustment of the volume measured by the assembly  130 .  
         [0023]     The processor  30 , which may include programs embedded in hardware and/or software, is adapted to produce a series of commands for controlling the operation of the system  20  and components thereof. During a first operation, passages in the components of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the gradient solution are purged of foreign materials. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  42  is in communication with port  44 . The processor  30  then commands the motor-driven gradient syringe  64  to pull the syringe plunger to aspirate a predetermined volume (e.g., 20 milliliters (ml)) of gradient solution from the gradient reservoir  62 . The volume of gradient solution aspirated into the syringe barrel should be sufficient to fill the passages in the components of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the gradient solution. Once an appropriate volume of gradient solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  44  is in communication with the outlet port  50  and the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  72  is in communication with port  78 . Once these connections are made, the processor  30  commands the motor-driven gradient syringe  64  to push the syringe plunger to dispense the held volume of gradient solution from the syringe to the waste reservoir  98  at a predetermined flow rate (e.g., 10 ml/minute). The flow rate of gradient solution dispensed from the syringe barrel is chosen so that the passages of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the gradient solution are rinsed with gradient solution.  
         [0024]     During a second operation, passages in the components of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the buffer solution are purged of foreign materials. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 20 ml) of buffer from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to fill the passages in the components of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the buffer solution. Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . The stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  remain in their prior positions so that port  72  is in communication with port  78 . Once these connections are made, the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe to the waste reservoir  98  at a predetermined flow rate (e.g., 35 ml/minute). The flow rate of buffer solution dispensed from the syringe barrel is chosen so that the passages of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the buffer solution are thoroughly rinsed with buffer solution without over-pressurizing the components.  
         [0025]     During a third operation, the rotor  24  of the ultracentrifuge  22  is filled with buffer solution. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 140 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to fill the rotor  24  of the ultracentrifuge and passages in the components of the solution delivery assembly  32 , and the sample delivery assembly  34  that carry the buffer solution, as well as passages in the components of the overflow management assembly  36  and those leading from this assembly to the waste reservoir  98 . Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . Further the processor  30  commands the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  72  is in communication with outlet port  80 , the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  to rotate so that port  112  is in communication with outlet port  120 , and the stopcocks  152 ,  154 ,  156  of the overflow management assembly  38  to rotate so that port  142  is in communication with port  148  leading to the waste reservoir  98 . Once these connections are made, the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe to the rotor  24  at a predetermined flow rate (e.g., a flow rate between about 5 ml/minute and about 35 ml/minute). The flow rate of buffer solution dispensed from the syringe barrel is chosen, so that the passages of the solution delivery assembly  32  and the sample delivery assembly  34  that carry the buffer solution are not over-pressurized. As will be appreciated by those skilled in the art, the operation of filling the ultracentrifuge rotor  24  with buffer solution may be accomplished using a smaller syringe by repeating the sequence of steps described above until a volume of buffer solution equal to that described above is delivered to the system components. For example, if a 100 ml capacity buffer syringe  68  were used and the volume of buffer solution needed was determined to be 140 ml, the syringe could be filled to 100 ml during a first sequence and 40 ml during a second sequence. Alternatively, the syringe could be filled to 70 ml during both sequences.  
         [0026]     During a fourth operation, the system is de-bubbled. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  the commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 80 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to compensate for an increase in rotor  24  size due to rotation and a loss in air. Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . The stopcocks  82 ,  84 ,  86  of the delivery assembly  34  remain in their prior positions so that port  72  is in communication with outlet port  80 , the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  remain in their prior positions so that port  112  is in communication with outlet port  120 , and the stopcocks  152 ,  154 ,  156  of the overflow management assembly  38  remain in their prior positions so that port  142  is in communication with port  148  leading to the waste reservoir  98 . Once these connections are made, the processor  30  commands the ultracentrifuge  22  to rotate the rotor  24  at a predetermined speed (e.g., 20,000 rpm). The processor  30  may also command the ultracentrifuge to use a predetermined final ramp speed (e.g., 100 rpm) and a predetermined coast speed (e.g., 500 rpm). As will be appreciated by those skilled in the art, when the ultracentrifuge  22  is accelerating, it initially accelerates at a slower rate (e.g., 4 rpm/second), and then a faster rate (e.g., 54 rpm/second). The final ramp speed is the speed at which the ultracentrifuge speeds up from the slower acceleration rate to the faster acceleration rate. Likewise, when the ultracentrifuge  22  is decelerating, it initially decelerates at a faster rate until it reaches the coast speed. The processor  30  may also activate a chiller surrounding the ultracentrifuge  22  to cool the components of the ultracentrifuge. When the rotor  24  reaches a predetermined intermediate speed (e.g., 5000 rpm), the processor commands the motor driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe to the rotor  24  at a predetermined flow rate (e.g., 5 ml/minute). The flow rate of buffer solution dispensed from the syringe barrel is chosen so that the solution is gently introduced without turbulence to reduce the potential for introduction of bubbles into the system. The solution introduced during this step compensates for air that is driven from the system and for an increase in rotor volume caused by its high rotational speed. The processor  30  maintains the rotor speed at the predetermined speed (e.g., 20,000 rpm) for a predetermined time (e.g., 3 minutes) selected so that the air is separated from the solution and driven out of the rotor. After the predetermined time, the processor commands the ultracentrifuge to brake.  
         [0027]     During a fifth operation, the rotor  24  of the ultracentrifuge  22  is partially filled with gradient solution. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  42  is in communication with port  44 . The processor  30  then commands the motor-driven gradient syringe  64  to pull the syringe plunger to aspirate a predetermined volume (e.g., 50 ml) of gradient solution from the gradient reservoir  62 . The volume of gradient solution aspirated into the syringe barrel should be equal to a predetermine portion of the ultracentrifuge rotor  24  (e.g., about half of the rotor volume). Once an appropriate volume of gradient solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  44  is in communication with the outlet port  50 . The stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  reman in their prior positions so that port  72  is in communication with the outlet port  80 , the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  remain in their prior positions so that port  112  is in communication with outlet port  120 , and the stopcocks  152 ,  154 ,  156  of the overflow management assembly  38  remain in their prior positions so that port  142  is in communication with port  148  leading to the waste reservoir  98 . Once these connections are made, the processor  30  commands the motor-driven gradient syringe  64  to push the syringe plunger to dispense the held volume of gradient solution from the syringe toward the rotor  24  at a predetermined flow rate (e.g., 10 ml/minute). The flow rate of gradient solution dispensed from the syringe barrel is chosen so that the solution is gently introduced without turbulence to reduce the potential for introduction of bubbles into the system.  
         [0028]     During a sixth operation, the gradient solution is chased with buffer solution so that substantially all of the gradient solution is in the rotor  24  of the ultracentrifuge  22 , and the rotor has the predetermined portion filled with gradient solution. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 20 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to displace all of the gradient solution in the passages between the solution delivery assembly  32  and the rotor  24 . The stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  remain in their prior positions so that port  72  is in communication with outlet port  80 , the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  remain in their prior positions so that port  112  is in communication with outlet port  120 , and the stopcocks  152 ,  154 ,  156  of the overflow management assembly  38  remain in their prior positions so that port  142  is in communication with port  148  leading to the waste reservoir  98 . Once these connections are made, the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe to the rotor  24  at a predetermined flow rate (e.g., 10 ml/minute). The flow rate of gradient solution dispensed from the syringe barrel is chosen so that the solution is gently introduced without turbulence to reduce the potential for introduction of bubbles into the system.  
         [0029]     The rotor  24  is now filled with buffer solution and gradient solution. During a seventh operation, the processor  30  commands the ultracentrifuge  22  to rotate the rotor  24  at a predetermined speed (e.g., 20,000 rpm). The processor  30  may also command the ultracentrifuge to use a predetermined final ramp speed (e.g., 3500 rpm) and a predetermined coast speed (e.g., 7000 rpm). The ramp speed and coast speed should be selected so that the solutions gently transition from horizontal separation to vertical separation and back. Thus, the solutions are not mixed as the rotor  24  accelerates to and decelerations from the predetermined speed. As the rotor  24  is accelerating to the predetermined speed, passages in the sample delivery assembly  34  are purged. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 40 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to fill corresponding passages in the sample deliver assembly  34  and the fraction collection assembly  36  as will be apparent below. Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . Further, the processor  30  commands the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  72  is in communication with outlet port  80 , and the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  to rotate so that port  112  is in communication with port  114 . Once these connections are made, the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe into the priming accumulator  134  at a predetermined flow rate (e.g., 35 ml/minute). Once the buffer solution is dispensed, the processor  30  commands the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  74  is in communication with outlet port  80 . Once these connections are made, the processor  30  commands the peristaltic sample pump  94  to run in reverse for a predetermined period (e.g., one minute) at a predetermined flow rate (e.g., 30 ml/minute) to draw the buffer material from the priming accumulator  134  so the sample delivery passages are primed.  
         [0030]     As the rotor  24  continues to accelerate to the predetermined speed, passages in the sample delivery assembly  34  used to recycle the sample solution are purged. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 40 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to fill corresponding the passages in the sample deliver assembly  34  and the fraction collection assembly  36  as will be apparent below. Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . Further, the processor  30  commands the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  72  is in communication with outlet port  80 , the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  to rotate so that port  112  is in communication with port  114 . Once these connections are made, the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe into the priming accumulator  134  at a predetermined flow rate (e.g., 35 ml/minute). Once the suffer solution is dispensed, the processor  30  commands the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  74  is in communication with outlet port  80 . Once these connections are made, the processor  30  commands the peristaltic recycle pump  96  to run in reverse for a predetermined period (e.g., one minute) at a predetermined flow rate (e.g., 30 ml/minute) to draw the buffer material from the priming accumulator  134  so the recycle passages are primed.  
         [0031]     During an eighth operation, additional buffer solution is added to compensate for rotor growth at speed. The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 35 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to compensate for rotor growth. Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . Further, the processor  30  commands the stopcocks  82 ,  84 ,  86  of the sample delivery assembly  34  to rotate so that port  72  is in communication with outlet port  80 , and the stopcocks  122 ,  124 ,  126  of the fraction collection assembly  36  to rotate so that port  112  is in communication with outlet port  120 . Once the rotor  24  reaches a predetermined intermediate speed (e.g., 5000 rpm), the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe to the rotor  24  at a predetermined flow rate (e.g., 5 ml/minute). The flow rate of buffer solution dispensed from the syringe barrel is chosen so that the solution is gently introduced without turbulence to reduce the potential for introduction of bubbles into the system.  
         [0032]     During an ninth operation, the sample solution is loaded into the ultracentrifuge rotor  24 . Once the rotor  24  reaches another predetermined intermediate speed (e.g., 20,000 rpm), the processor  30  commands the sample delivery assembly manifold stopcocks  82 ,  84 ,  86  to rotate so that port  74  is in communication with outlet port  80 . Once these connections are made, the processor  30  commands the peristaltic sample pump  94  to run in a forward direction at a predetermined flow rate (e.g., 10 ml/minute) to dispense sample solution into the ultracentrifuge rotor  24 . After a predetermined period (e.g., two minutes), the processor  30  commands overflow management assembly manifold stopcocks  152 ,  154 ,  156  to rotate so that port  142  is in communication with port  146 . This allows solution leaving the ultracentrifuge rotor  24  (i.e., eluent) to be collected in the recycle reservoir  102 .  
         [0033]     During a tenth operation, the sample solution is chased with buffer solution so that substantially all of the sample solution enters the rotor  24  of the ultracentrifuge  22 . The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to a predetermined volume (e.g., 80 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to displace all of the sample solution in the passages between the sample delivery assembly  34  and the rotor  24 . Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . When the sample reservoir  100  is empty, the processor  30  commands the sample delivery assembly manifold stopcocks  82 ,  84 ,  86  to rotate so that port  72  is in communication with outlet port  80 . Further, the processor  30  stops the peristaltic sample pump  94 , and the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe toward the rotor  24  at a predetermined flow rate (e.g., 5 ml/minute). The processor  30  then commands the ultracentrifuge to increase the rotor speed to a predetermined separation speed (e.g., 35,000 rpm).  
         [0034]     After a predetermined period (e.g., eight minutes) at the separation speed, an eleventh operation is commences in which the collected eluent is recycled through the ultracentrifuge rotor  24 . The processor  30  commands the overflow management assembly manifold stopcocks  152 ,  154 ,  156  to rotate so that port  142  is in communication with port  148  so that the rotor output is sent to the waste reservoir  98 . Further, the processor  30  commands the sample delivery assembly manifold stopcocks  82 ,  84 ,  86  to rotate so that port  76  is in communication with outlet port  80 . Once these connections are made, the processor  30  commands the peristaltic recycle pumps  96  to run in a forward direction at a predetermined flow rate (e.g., 10 ml/minute) to draw the recycled eluent from the recycle reservoir  96 . After the recycle pump  96  has been running for a predetermined period (e.g., four minutes), the processor  30  commands the overflow management assembly manifold stopcocks  152 ,  154 ,  156  to rotate so that port  142  is in communication with the outlet port  150  so that the rotor output is sent to the final flow reservoir  160 . The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . The processor  30  then commands the motor-driven buffer syringe  68  to pull the syringe plunger to aspirate a predetermined volume (e.g., 80 ml) of buffer solution from the buffer reservoir  66 . The volume of buffer solution aspirated into the syringe barrel should be sufficient to displace all of the sample solution in the passages between the sample delivery assembly  34  and the rotor  24 . Once an appropriate volume of buffer solution is aspirated, the processor  30  commands the stopcocks  52 ,  54 ,  56  of the solution delivery assembly  32  to rotate so that port  48  is in communication with the outlet port  50 . When the recycle reservoir  102  is empty, the processor  30  commands the sample delivery assembly manifold stopcocks  82 ,  84 ,  86  to rotate so that port  72  is in communication with outlet port  80 . Further, the processor  30  stops the recycle pump  94 , and the processor  30  commands the motor-driven buffer syringe  66  to push the syringe plunger to dispense the held volume of buffer solution from the syringe toward the rotor  24  at a predetermined flow rate (e.g., 5 ml/minute). After a predetermined period (e.g., four minutes), the processor  30  commands the overflow management assembly manifold stopcocks  152 ,  154 ,  156  to rotate so that port  142  is in communication with port  148  so that the rotor output is sent to the waste reservoir  98 . The processor  30  commands the solution delivery assembly manifold stopcocks  52 ,  54 ,  56  to rotate so that port  48  is in communication with port  46 . After a predetermined separation period (e.g., two hours), the processor  30  stops the ultracentrifuge rotor rotation. As the ultracentrifuge rotor  24  stops rotating, less buoyant particles move to the bottom of the rotor and more buoyant particles move to the top of the rotor  
         [0035]     During a twelfth operation, particles are removed from the bottom of the rotor  24 . As will be appreciated less buoyant particles will be removed before more buoyant particles. The processor  30  commands the motor driven stage of the fraction collector  132  to move to a home position. The processor  30  then commands the lower stopcock  176  to rotate so port  182  is only connected to port  186 , the upper stopcock  178  to rotate so port  190  is only connected to port  192 , and the stopcock  152  of the overflow management assembly manifold  140  to rotate so port  142  is connected to port  144 . This configuration results in pressure equalization in the ultracentrifuge rotor  24  and the central tube  170  of the fraction volume measurement assembly  130 .  
         [0036]     Once the pressures are equalized, the processor  30  commands the stage of the fraction collector  132  to align a first well of the collection receptacle so solution traveling through port  186  of the lower stopcock  176  will fall into the first well. When the first well is in position, the processor  30  commands the lower stopcock  176  to rotate so port  182  is only connected to port  184 , and separated solution drains from the ultracentrifuge rotor  24  into the central tube  170  of the fraction volume measurement assembly  130 . As the solution rises in the tube  170 , it eventually crosses the level of the predetermined infrared transmitter and receiver (i.e., either  200 ,  204  or  202 ,  206 ) indicating a predetermined volume (e.g., 1 ml or 2 ml) is in the tube. When this level is detected, the processor  30  commands the lower stopcock  176  to rotate so port  182  is only connected to port  186  and the upper stopcock  178  to rotate so port  194  is only connected to port  190 , allowing the source  198  to gently push the solution in the tube  170  into the well in the fraction collector receptacle. After a sufficient dwell time (e.g., two seconds), the processor  30  commands the upper stopcock  178  to rotate so port  194  is only connected to port  192 , and commands the stage of the fraction collector  132  align a second well of the collection receptacle so solution traveling through port  186  of the lower stopcock  176  will fall into the second well. The measurement and dispensing steps are repeated until a predetermined number of wells (e.g., 96 wells) is filled with solution. After the predetermined number of wells is filled, the processor commands the stage of the fraction collector  132  to align a waste well of the collection receptacle so solution traveling through port  186  of the lower stopcock  176  will fall into the waste well. The measurement and dispensing steps are repeated until a level is not detected, indicating the rotor  24  is empty.  
         [0037]     When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.  
         [0038]     As various changes could be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.