Abstract:
A method for control of the drive means for a reciprocating piston pump delivering liquid to a spring loaded piston liquid accumulator providing high volume compliance whereby the accumulator liquid volume is controlled within narrow limits by continuous control of power to the pump drive motor. The accumulator achieves high volume compliance by arranging the kinematics of a main spring loading the accumulator piston to have a negative spring rate equal to the sum of all other positive spring rates produced by a second spring used to adjust the accumulator pressure, by the diaphragm (piston), and by a flexure support for a sensor lever. This sensor lever moves with the accumulator diaphragm to actuate an optical sensor producing an electric signal indicative of small changes in liquid volume in the accumulator. This signal in turn continuously modulates the power to the motor driving the pump so as to maintain the accumulator liquid volume close to a datum value during a large portion of the pump delivery cycle. This close control of liquid volume in a high compliance accumulator provides substantially constant pulse free pressure liquid delivery from a pulsatile pump. The second spring may be adjusted to modify this constant pressure without disturbing the balance between positive and negative spring rates. Adjustment may be manual or automatic in response to liquid temperature whereby liquid pressure is automatically increased with lower liquid temperatures to compensate for increased liquid viscosity to maintain liquid flow substantially constant through an apparatus such as flow cytometry used for particle analysis or particle sorting.

Description:
This is a division of Ser. No. 09/224,405 filed Dec. 31, 1998 which is a division of Ser. No. 08/779,505 filed Jan. 7, 1997 now U.S. Pat. No. 5,915,925 issued Jun. 29, 1999. 
    
    
     BACKGROUND 
     1. Field of Invention 
     This invention relates to a system for delivering pressurized liquid to an apparatus, such as a flow cytormeter, and in particular to a system having an improved liquid accumulator/pump control means for providing continuous modulation of power to a liquid pump drive means. 
     2. Description of Related Art 
     Flow cytometry apparatus has commonly used a liquid suspension of particles ensheathed in a particle-free liquid wherein this coaxial flow is passed through an analysis region and thence often to a particle sorting means. Such coaxial flow systems are shown in an article by P. J. Crossland-Taylor, Nature 171, 37 (1953) and in U.S. Pat. No. 3,826,364, which are hereby referred to and incorporated herein. Sheath liquid is usually a phosphate buffered saline solution and is usually supplied to the analysis region from a closed reservoir pressurized by air from an air pressure regulator connected to a source of air at higher pressure (note items 16, 26, and 22 of U.S. Pat. No. 3,826,364). Since particle analyzers and particle sorters often depend on consistent liquid flow velocities through the analysis region, this air pressurized sheath supply system has the following shortcomings: 
     1) As the sheath supply reservoir empties during operation of the flow cytometer the liquid level decreases and the loss of head causes a decrease in liquid flow rate; 
     2) Changes in sheath liquid temperature cause changes in sheath liquid flow rate due to changes in liquid viscosity. Changes in liquid temperature can result from changing ambient air temperature or from sheath reservoir replenishment with liquid at a different temperature. 
     3) Replenishment of sheath liquid is inconvenient, requiring stopping operation of the flow cytometer, de-pressurizing the reservoir, opening and refilling the reservoir, re-pressurizing the reservoir and restarting the flow cytometer; 
     4) The pressurized reservoir has often been a stainless steel ASME pressure vessel which is both expensive and unsuitable for visual observation of liquid level in the reservoir; 
     5) Air dissolves in the sheath liquid in time and can later be released as bubbles as the liquid loses pressure while flowing through filters, valves, and conduits to the analysis and sorting regions. Bubbles in these regions often prevent proper analysis or sorting functions; and 
     6) When pressureized air supply is not available at a flow cytometer installation, then a separate air compressor, motor, reservoir, and controls must be provided. 
     Attempts to use gear or centrifugal pumps to pressurize sheath liquid, usually phosphate buffered saline, have not produced practical designs. Neither pump is inherently self-priming so initial start up or restart after running out of liquid requires the operator to perform special procedures such as bleeding air from the system. If either pump is kept running when liquid flow through the flow cytometer stops, then the pump will tend to overheat and be damaged. Solutions such as an overflow/overpressure line for returning pressurized liquid back to the supply reservoir or stopping the pump add cost and complexity. Also gear and centrigugal pumps suitable for long life operating with corrosive saline are expensive. 
     Many of these shortcomings of gear or centrifugal pumps are avoided by diaphragm pumps, particularly those with polymer housings and with elastomer diaphragms and check valves. However, diaphragm pumps require a liquid accumulator to supply pressurized liquid during the refilling stroke of the pump. Common accumulators employ a piston loaded by a spring or a bladder loaded by compressed gas or combinations thereof (as is shown in U.S. Pat. No. 4,278,403. This patent shows an accumulator  35  which operates a pump P via a switch  43  in an on/off mode from a pressure movable partition element, piston  36 . This on/off mode of pump control with its dead band between On and Off conditions results in significant changes in pressure in accumulator  35 . In addition the friction from seals for piston  36  and stem  41  produce inaccuracies in the sensing of pressure in accumulator  35 . Also these seals are subject to wear and leakage which limit the durability of accumulator  35 . 
     OBJECTS AND ADVANTAGES 
     Accordingly several objects and advantages of my invention are: 
     a) to provide essentially pulse-free constant pressure liquid to an apparatus, such as a flow cytometer, unaffected by liquid level changes in the supply reservoir; 
     b) to employ an unpressurized supply reservoir which is easy to refill, is simple and low cost, may be raised and lowered without affecting the liquid supply pressure, does not introduce air into the sheath liquid, does not require a separate air supply and valves, may be sized large to reduce replenishment frequency, may be transparent for visual observation of liquid level, and may be replenished without stopping operation of the apparatus; 
     c) to provide for manual or automatic adjustment of the sheath liquid pressure as required to compensate for variations in sheath liquid temperature and thereby maintain sheath liquid flow rate substantially constant and thus maintain critical flow cytometer timing such as: 
     Particle transit time from laser beam to drop break-off for drop-in-air sorters, 
     Particle transit time from laser beam to catcher tube for a catcher tube sorter; 
     Particle transit time between laser beams in a multiple laser analyzer or sorter; 
     d) to provide a novel liquid accumulator which can accept the liquid volume delivered by one stroke of a diaphragm pump with negligble change in liquid pressure; and 
     e) to provide a self-priming pump in a liquid supply system where the liquid contacts only non-metal parts thus avoiding metal corrosion and contamination of the liquid. 
     Other objects and advantages are to provide apparatus and method for delivering pressurized liquid to an apparatus which is small, simple, low cost, reliable, durable, quiet, accurate, essentially pulse-free, and which operates with low electric power. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a pulse-free, constant pressure liquid delivery system which may be adapted for connection to an apparatus, such as a flow cytometer. Preferred embodiments of the invention provide for manual or automatic adjustment of the liquid pressure to compensate for variations in liquid supply temperature and thus maintain constant liquid flow rate through the apparatus. This invention avoids many of the problems, inconveniences, and cost associated with other liquid supply systems by use of a novel liquid accumulator design and an electric motor driven reciprocating diaphram pump controlled by a simple electronic control responsive to the liquid volume in the accumulator. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The invention may be understood, and further advantages and uses are more readily apparent, when considered in view of the following detailed description of the exemplary embodiments, taken with the accompanying drawings, in which: 
     FIG. 1 is a simplified schematic diagram of the liquid supply system as connected to a flow cytometer; 
     FIG. 2 is a cross-sectional view of the liquid supply assembly taken along the line  2 — 2  of FIG. 3; 
     FIG. 3 is a plan view of the volume sensor; 
     FIG. 4 is a cross-sectional view of the manual pressure adjustment; 
     FIG. 5 is a diagram of the kinematic features of the invention; 
     FIG. 6 is an electrical diagram of the volume sensor, volume control, and pump motor. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Referring now to the drawings and to FIG. 1 in particular, there is shown a simplified schematic diagram of liquid supply system  10  supplying flow cytometer  12  with a constant liquid flow, all constructed according to the teachings of the invention. Liquid supply system  10  includes reservoir  20  connected via conduit  34  to pump  22 . thence via conduit  36  to accumulator assembly  30 , thence via conduit  38  to the inlet of flow cytometer  12 . Accumulator assembly  30  is part of liquid supply control means  32  for supplying a constant flow of liquid to flow cytometer  12  which includes pump  22 , pump motor  24 , volume sensor  28 , volume control  26 , and adjustment actuator  18 . Liquid supply system  10  further includes adjustment control  16  connected to liquid temperature sensor  14  and adjustment actuator  18 . 
     Referring now to FIG. 2 there is shown a cross-sectional view (taken along the line  2 — 2  of FIG. 3) through the middle of liquid supply assembly  32  which shows accumulator assembly  30  including diaphragm  54  clamped between diaphragm retainer  56  and accumulator body  52  by screws  58 , spacers  60 , main support  40 , and nuts  62 . Diaphragm  54  is of the constant area rolling diaphragm type such as for example are manufactured by Bellofram Corporation. Diaphragm  54  is connected via piston  88 , ball  90 , diaphragm screw  92 , and nut  94  to sensor lever  42 . Sensor lever  42  is pivotably supported by flexure pivot  44  clamped to sensor lever  42  by flexure retainer  46 , two screws  48 , and two nuts  50  and is clamped to main support  40  by flexure retainer  47 , two screws  49 , and two nuts  51 . A main spring  64  is attached to sensor lever  42  by main spring support screw  66  and nut  68  and is attached to main support  40  by main spring support screw  67  and nut  69 . Main spring  64  is a helical extension spring with a hook at each end for insertion in a hole in spring support screws  66  and  67 . An adjustment spring  70  is attached to sensor lever  42  by adjustment spring support screw  72  and nut  74  and to adjustment actuator  18  by coupling  76  and set screw  78 . Adjustment spring  70  is a helical extension spring with a hook at each end for insertion in a hole in spring support screw  72  and in coupling  76 . Adjustment actuator  18  is fastened to main support  40  by two spacers  82 , two screws  84  and two nuts  86 . The adjustment actuator  18  may be, for instance, a stepper motor driven linear actuator such as manufactured by Haydon Switch and Instrument and sold as Model No. 26541 which has 0.001 inch motion per electrical steo. Pump  22  and pump motor  24  may be an integrated assembly such as is manufactured by KNF Neuberger as Model No. NF30KVDC or NF1.30KVDC which are rated for continuous pumping at pressures up to 15 and 85 psi respectively. Pump motor  24  and volume sensor  28  are connected electrically to volume control  26 . Pump  22  with pump motor  24  may be mounted on the main support  40  or elsewhere. Liquid supply assembly  32  is preferrably oriented so air is naturally purged from accumulator assembly  30  and pump  22  when liquid flows through these components during start up. 
     Referring now to FIG. 3 there is shown a plan view of sensor lever  42  and volume sensor  28  which is fastened to main support  40  by screws  98  and two nuts  100 . Volume sensor  28  comprises an infrared light emitting diode facing an NPN silicon phototransistor encased in a black thermoplastic housing such as for example is manufactured and sold by Honeywell as Model No. HOA0890-T51. The reduced width end of sensor lever  42  is located within a slot between the light emitting diode and the phototransistor. 
     Referring now to FIG. 4, there is shown a cross-sectional view of the manual pressure adjustment where the adjustment actuator  18 , coupling  76 , and set screw  78  have been replaced by support plate  102 , and manual adjustment  104  which is threadably engaged with manual adjustment coupling  106  which has a square cross-section slidably engaged with main support  40  in a square hole to prevent rotation of manual adjustment coupling  106 . 
     Referring now to FIG. 5 there is shown a diagram of the essential kinematic features of the invention provided to facilitate explanation of the design of an accumulator having substantially infinite volume compliance. Volume compliance is de fined as a small change in accumulator volume divided by the resulting change in accumulator pressure. A,B,C&amp;D are dimensions from the flexure pivot  44  pivot point P to the centerlines for the forces from main spring  64 , adjustment spring  70 , diaphragm  54 , and the light beam of volume sensor  28  respectively. H is the height above pivot point P of the contact of main spring  64  with main spring support screw  66 . a is the angle defined by tan a=H/A. L is the installed length of main spring  64  between contacts with main spring support screws  66  and  67 . The following terms are defined here: 
     K M  is the spring rate of main spring 64—lbs./inch 
     K A  is the spring rate of adjustment spring 70—lbs./inch 
     K D  is the spring rate of diaphragm 54—lbs./inch 
     T P  is the torsional spring rate of flexure pivot  44  as installed, in inch lbs./radian. defined as the rate of change in moment about pivot point P per radian change in angle a due to motion of sensor lever  42  about pivot point P 
     A D  is the effective area of diaphragm  54  exposed to liquid pressure—square inches 
     P s  is the liquid pressure acting on the diaphragm—psi 
     F M  is the tension force of main spring 64—lbs. 
     F A  is the tension force of adjustment spring 70—lbs.                Accumulator                 Compliance               (     cubic                   inch   /   psi       )           =       A   D   2         K   D     +       K   A            B   2         C   ~     2         +       T   p         C   ~     2       +       K   M            A   2       C   2         -       F   M            (     L   -   H     )     L          H       C   ~     2                                    
     Referring now to FIG. 6 there is shown a typical volume control  26  used with volume sensor  28  and pump motor  24 . Typical components are IRF520 N channel MOSFET, R 1 =560 ohm and R 2 =470,000 ohm. 
     Referring again now to FIG. 1, in operation there will be liquid flow from the supply reservoir  20  through conduit  34  to pump  22  and then through conduit  36  to accumulator assembly  30  and then through conduit  38  to flow cytometer  12 . Pump  22  is driven by pump motor  24  which is controlled by volume control  26  which is responsive to volume sensor  28 . Referring now to FIG. 2, there is shown that as less liquid is contained in accumulator assembly  30  diaphragm  54  and piston  88  move towards accumulator body  52 . In turn ball  90 , diaphragm screw  92 , nut  94 , and sensor lever  42  also move towards accumulator body  52 . Sensor lever  42  then moves to permit more light from the light emitting diode to reach the phototransistor in volume sensor  28  which increases the phototransistor conductivity. As shown on and now referring to FIG. 6, this increases the voltage between the gate G and the source S of the MOSFET which increases the current through pump motor  24  driving pump  22 . Pump  22  then increases its discharge of liquid into accumulator assembly  30  which causes diaphragm  54 , piston  88 , ball  90 , diaphragm screw  92 , nut  94 , and sensor lever  42  to move away from accumulator body  52 . This motion of sensor lever  42  reduces the light from the light emitting diode reaching the phototransistor in volume sensor  28  which reduces its conductivity. This decreases the voltage between the gate and the source of the MOSFET which reduces the current (and the torque) through the pump motor  24  driving pump  22 . Pump  22  then slows down or stops delivering liquid to accumulator assembly  70 . In this manner, balance is obtained in this closed-loop control system. This balance is obtained both statically and dynamically throughout the delivery stroke of pump  22  from bottom dead center to near top dead center. However, near the top dead center the control loop tends to become unstable and a small fraction of the stroke volume of pump  22  is delivered to accumulator assembly  30  whether needed or not needed. This fraction is typically less than 10% of the stroke volume of pump  22 . After passing top dead center, pump  22  refills from reservoir  20  and returns to bottom dead center rapidly for continued control of liquid volume within accumulator assembly  30 . 
     It is clear that accumulator assembly  30  will have small but significant volume changes during each delivery cycle of pump  22 . the liquid supply assembly  32  is provided with a novel kinematic design so that supply pressure P s  within accumulator assembly  30  is essentially unaffected by these small liquid volume changes whereby P s  is fixed within less than + or −0.1% fluctuation during continued operation of pump  22 . This accumulator function is produced by arranging the main spring  64  kinematically to produce a negative spring rate at diaphragm  54  which numerically equals the positive spring rate at diaphragm  54  produced by the sum of the spring rates of diaphragm  54 , adjustment spring  70  and flexure pivot  44 . Referring now to FIG. 5 the negative spring rate effect of main spring  64  is produced by making H large enough in relation to the other parameters that as sensor lever  42  moves to increase angle a, the fractional decrease in moment arm from main spring force to pivot P is greater than the fractional increase in force from the main spring due to its greater extension. The net effective spring rate, K e , at the diaphragm centerline is given by:                K   e     =       K   D     +       K   A            B   2       C   2         +       T   p       C   2       +       K   M            A   2       C   2         -       F   M            (     L   -   H     )     L          H     C   2         -     lb   /     in   .                 Eq   .              1                               where 
                     T   p     =         Ewt   3       12                 h          inch                     lbs   .     /   radian               Eq   .              2                                inch lbs./radian  Eq. 2 
     where for pivot support  44 : 
     E=Young&#39;s modulus of elasticity—psi 
     w=width—inches 
     t=thickness—inches 
     h=height—inches 
     The volume compliance, C volume , of accumulator assembly  30  is: 
     
       
         C volume   
       
     
                     C   volume     =         A   D   2       K   e       -       in        .   3       /   psi               Eq   .              3                                in. 3 /psi  Eq. 3 
     Infinite compliance for small changes in liquid volume is obtained when K e =0. By setting K e =0 in Eq. 1 and solving for H we obtain:              H   =       L   ≠         L   2     -     4                 m           2             Eq   .              4                               where 
                   m   =       L     F   M            (         K   D          C   2       +       K   A          B   2       +     T   p     +       K   M          A   2         )               Ew   .              5                                  Ew. 5 
     Eq. 4 provides two solutions for H. Each positive, real solution is valid and may be used. Where two valid solutions exist the smaller H is preferred since it results in a more compact liquid supply assembly  32 . 
     The liquid supply pressure, P s , in accumulator assembly  30  is:                P   s     =             F   M          A   C       +       F   A          B   C       +     F   D     +       (       T   p        x                 change                 in                 a     )     C         A   D       -   psi             Eq   .              6                                
     Normally diaphragm  54  and flexure pivot  44  are undeflected from their relaxed positions and thus F D  and change in a are nearly zero and may usually be neglected. Eq. 6 then becomes:                P   s     =             F   M          A   C       +       F   A          B   C           A   D       -   psi             Eq   .              7                                
     F M  is set to give the desired minimum value of P s  when F A  is zero. H is calculated from this value of F M  using Eqs. 4 and 5. Then F A  is calculated to give the maximum value of P s . 
     F A  is adjusted by varying the extension of adjustment spring  70  by linear motion produced by adjustment actuator  18  which moves one end of adjustment spring  70  through coupling  76 . The other end of adjustment spring  70  is supported at a location opposite Pivot P so that negligible changes in moment arm B occur with small changes in angle a. Thus there is no significant change in total spring rate K e  as F A  is varied from minimum to maximum. The spring rate of adjustment spring  70  is chosen so the desired adjustment range of P s  can be obtained with the available linear motion of adjustment actuator  18 . For the preferred embodiment the available motion is about 0.500 inch with 0.001 inch per step of the stepping motor. For an adjustment range of 0-50% of P s  each step therefore produces about 0.1% change in P s . This provides fine control of P s  setting. 
     When used with flow cytometer  12  the liquid supply system  10  is usually operated so as to increase P s  as liquid temperature entering the flow cytometer  12  decreases to compensate for the effects of increased liquid viscosity and thus maintain constant both liquid flow and velocity through flow cytometer  12 . Constant liquid velocity allows for fixed settings for delay time in drop-in-air and catcher tube sorters as well as the transit time for cells passing between laser beams in a cell analyzer. Liquid temperature sensor  14  provides a signal to adjustment control  16  which then sends the appropriate number of electrical step signals to adjustment actuator  18  to drive it from a home or fixed starting position to the desired compensated operating position and thus apply the required extension to adjustment spring  70  to obtain the required supply pressure P s . Adjustment control  16  may use an EPROM or other suitable memory device to accomplish the function of a look-up table of stepper motor steps versus fluid temperature. The adjustment control  16  has conventional electronics suitable for driving the stepper motor of the adjustment actuator  18 . The adjustment control  16  may be implemented in various ways by those skilled in the art and is therefore not described in more detail here. 
     While the adjustment control  16  is shown as responsive to liquid temperature it is obvious that it could be responsive to any suitable operating parameter of flow cytometer  12  which can be sensed to provide either a closed loop control of that parameter or a programmed bias of P s  produced in response to that parameter. Such parameters may be, for example, liquid flow as sensed by the transit time for a particle to pass through two laser beams or particle velocity as sensed by the time duration of a signal produced by a particle passing through the analysis region. A liquid flow parameter may also be sensed by the pressure drop across an orifice through which the liquid flows. 
     When such programmed or automatic control of P s  is not required, P s  may be adjusted by the apparatus shown in FIG.  4 . The manual adjustment  104  is supported by support plate  102  and is threadably engaged with manual adjustment coupling  106  which is prevented from rotating by having a square cross-section slidably engaged in a square hole in main support  40 . As manual adjustment  104  is rotated, manual adjustment coupling  106  moves linearly to change the extension of adjustment spring  70 . This changes its force, F A , which in turn changes P s  as set forth in Eq. 7. With a 32 thread per inch thread and a 50% change in P s  with a 0.500 inch motion of manual adjustment coupling  106 , there is about a 3% change in P s  for each revolution of manual adjustment  104 . 
     In conclusion, it can be readily understood that liquid supply system  10 , constructed according to the teachings of the invention provides a simple, compact, and economical apparatus for providing pulse-free pressurized liquid having no additional dissolved air at a pressure which is independent of liquid level in the supply reservoir, wherein this pressure may be adjusted manually or automatically to compensate for liquid temperature changes to provide for constant liquid flow and constant velocity of particles passing through a flow cytometer analysis and/or sorting region(s). 
     While the above description contains many specifications, these should not be construed as limitations on the scope of the invention, but rather as an example of one preferred embodiment of the invention. Many other variations are possible without departing from the teachings of the invention, of which a few alternatives will now be described: 
     The diaphragm pump  22  could be replaced by a peristaltic tubing pump or any other pump with suitable characteristics. The volume sensor  28  could be replaced with any non-contact proximity sensor such as for instance eddy current or capacitive devices. The accumulator  30  could use an unconvoluted or flat diaphragm. The adjustment actuator  18  could be replaced by any suitable electromechanical device such as for instance a rotary stepper motor driving a pinion gear coupled to a gear rack. It is also felt that adjustment actuator  18 , liquid temperature sensor  14 , and adjustment control  16  could be replaced by a non-electric means for adjustment of liquid pressure such as for instance a liquid thermal expansion apparatus. A sealed stainless steel bellows containing a liquid possessing a high thermal volume expansion characteristic could be placed in and exposed to the liquid passing through the accumulator body. One end of the bellows would be disposed in contact with the accumulator body. The other end of the bellows would be disposed so as to contact a compression spring interposed between the bellows and the accumulator diaphragm. In operation, as liquid temperature increases the liquid in the bellows expands, the bellows extends, the spring is further compressed, and the increased force on the diaphragm produces a decreased regulated liquid pressure P s . Flexure pivot  44  may be replaced with any suitable low frivtion bearing such as a ball bearing. A KNF Neuberger NF30KVDC pump which is rated for 15 psi is selected for the pump  22  and pump motor  24  combination in the preferred embodiment. For higher pressures a KNF Neuberger NF1.30KVDC pump which is rated for 85 psi continuous operation may be substituted. Both pumps are manufactured by KNF, Neuberger, Inc. of Trenton, N.J. Higher regulated liquid pressures may be obtained by the use of a smaller area diaphragm or by higher force main and adjustment springs in accumulator  30  without increasing the size of the liquid supply assembly  32 .