US3487938A - Chromatographic column head space reducer - Google Patents

Description

9 19m .1, A. MWmE-mm CHROMATOGRAPHIC COLUMN HEAD SPACE EEDUCER 2 Sheets-Sheet 1 Filed Aug. 5, 1968 N w OR- T m N E w T mm P J A W S E M m Ml EATTORNEYS mmw 1970 J A. PATTEEIWSEOM 5F7V93 CHROMATOGRAPHIC COLUMN HEAD SPACE HEDUCER Filed Aug. 8, 1968 2 Sheets-Sheet 2 BUFFER FLOW SAMPLE BNJECTOR FILTER IN VENTOR.

I JAMES A. PATTERSON BY 56-. 5 "70 m! WW ATFORNEYS United States Patent 3,487,938 CHROMATOGRAPHIC COLUMN HEAD SPACE REDUCER James A. Patterson, Los Altos, Califi, assignor t9 Sondell Research & Development Co., Palo Alto, Callf., a corporation of California Filed Aug. 8, 1968, Ser. No. 752,103

Int. Cl. B01d /08 US. Cl. 210-198 15 Claims ABSTRACT OF THE DISCLOSURE An automatic head space reducer for chromatographic columns is formed from cylinder means positioned over and fixed relative to the chromatographic column for receiving fluid under pressure from the fluid line, and piston means positioned within the head space of the chromatographic column slidably engaging the cylinder means. The piston is slidable within the head space, in response to fluid pressure transmitted through the fluid line and cylinder means, to minimize the head space and rest with a predetermined minimal differential force against chromatographic filter material retained in the column, while delivering fluid to the filter material from the fluid line through a channel in the piston.

This invention relates to a new and improved automatic head space reducer for chromatographic columns for eliminating the head space above chromatographic filter material retained in a chromatographic column through which the sample must be introduced.

Chromatographic columns generally comprise an elongated cylindrical housing having chromatographic filter material retained therein and a head space within the housing above the filter material through which a sample must be introduced. In order to provide a sharp chromatogram of the sample it is necessary that the sample be initiated into the column of chromatographic filter material in as narrow a zone as possible. It is thus desirable that the entire sample he introduced simultaneously across the top of the column of filter material. If there is any free space or head space above the filter material during loading of a sample, a concentration gradient, pH gradient or other gradient may be established across the bufler solution in the head space. The resulting dilution of the sample results in a delayed introduction of the sample into the filter material thereby destroying the sharpness of the resulting chromatogram. Eflicient and high resolution column chromatography thus requires that the head space above the column of filter material be minimized.

According to present techniques for minimizing the head space above the filter material in a chromatographic column, porous plugs or head spacers are manually positioned in the head space. Such manual head spacers may be screw-adjusted or otherwise manually adjusted against the top of the column of chromatographic filter material. By such manual placement, an unknown amount of physical pressure is placed against the filter material. Chromatographic filter materials such as cross-linked polymer resin beads are sensitive to such applied pressure. Thus, the osmotic or absorptive activity of the surface of the resin or other filter material may be adversely afiected by the pressure and thereby interfere with the sharpness and resolution of the resulting chromatograph. Furthermore, such manual placement may create pressures beyond the rupture limit of the system.

During gradient elution or elution of successive samples of varying concentration in chromatographic columns of the type using copolymer resin beads as a chromatographic filter material, the resin beads expand and contract,

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changing volume under the varying solvation stresses to which the beads are subjected. In systems wherein a porous plug is manually positioned in the column head space, the change in volume of the resin beads under the plug may subject the resin beads to physical pressures which destroy resolution of the chromatogram or which exceed the rupture limit of the system. To correct for the changing volume of the resin beads due to changing solvation stresses, the plug must be continua-11y manually adjusted, and even then with only limited accuracy.

It is an object of the present invention to provide a new and improved automatic head space reducer for minimizing head space in chromatographic columns by the application of predetermined and controlled pressures, thereby avoiding application of adverse or unknown physical pressure to the filter material or creation of harmful pressures in the fluid system.

Another object of the invention is to provide an automatic head space reducer which displaces head space in chromatographic columns by providing a movable piston which rests on the column of chromatographic filter material with a predetermined minimal differential force and which automatically follows the filter bed with the predetermined minimal differential force during change of volume of the column of chromatographic filter material.

A further object of the invention is to utilize the pressure in the fluid buffer line of the chromatographic system for automatically displacing the free head space above the filter material.

In order to accomplish these results the present invention contemplates providing a cylinder or pressure chamber positioned over and fixed relative to the chromatographic column. The cylinder or chamber includes an inlet for receiving fluid under pressure, an outlet to expel received fluid, and at least one channel for receiving and actuating a piston. One end of a piston is positioned slidably within the cylinder while the other end is positioned within the head space of the chromatographic column above the filter material and slides within the column to displace the head space in response to fluid pressure in the cylinder.

According to one aspect of the invention, the sample fluid and bufier solution are fed under pressure into the cylinder. The resulting pressure drives the piston downward to displace the head space over the chromatographic filter material. Upon leaving the cylinder, the fluid enters a passageway in the piston, applying pressure on a pressure release valve also formed in the piston. When the piston is urged against the chromatographic filter material, the valve opens at a predetermined differential force to release the sample and buifer at the head of the column of chromatographic filter material. The pressure exerted on the filter material is thereby controlled to a minimal level. Pressure from the sample and butfer line is utilized to displace the piston into the head space, the sample and buffer subsequently being released through the piston onto the chromatographic filter material upon development of a predetermined minimal differential force downward against the piston pressure release valve.

According to another aspect of the invention the cylinder comprises a separate pressure chamber having an inlet and outlet for receiving and discharging bulfer fluid under pressure from the fluid line for driving the piston. The piston is designed to rest with a predetermined minimal differential force against the chromatographic filter material despite changes in back pressure and changes in volume of the filter material. The operative differential force is the difference between the force downward on the piston from the pressure in sample and buffer fluid line and the force upward due to pressure upward on the piston from the chromatographic column. The piston is provided with a separate longitudinal passageway therethrough for receiving and delivering the sample fluid received from a sample injector and butter solution from the fluid line to the chromatographic column. The walls of the housing for the chromatographic column may be utilized to form part of the pressure chamber. In each case the piston is provided with an O-ring seal for sealing engagement with the walls of the housing of the chromatographic column. The invention also contemplates a piston design which absorbs the shock or pulses introduced into the fluid line by a positive displacement pump without transmitting the pressure pulses to the filter material in the chromatographic column.

Other objects, features and advantages of the present invention will become apparent in the following specification and accompanying drawings.

FIG. 1 is a side cross-sectional view of an automatic head space reducer embodying the present invention with the piston and head spacer disassembled.

FIG. 2 is a side cross-sectional view of another form of automatic head space reducer embodying the present invention with the piston and head spacer disassembled.

FIG. 3 is a side cross-sectional view of another automatic head space reducer embodying the present in vention.

FIG. 4 is a block diagram of the fluid system for a chromatographic column and any of the automatic head space reducers illustrated in FIGS. 1 through 3.

FIG. 5 is a side view of a form of automatic head space reducer embodying the present invention in which the sample and buffer solution are used to drive the piston directly.

FIG. 6 is a disassembled side cross-sectional view of the automatic head space reducer illustrated in FIG. 5. FIG. 6a is a side cross-sectional view of the piston.

In the embodiment of the present invention illustrated in FIG. 1 there is provided a cylinder 10, providing a pressure chamber, fixed to a mounting 11 positioned over and stationary relative to a chromatographic column 12. The chromatographic column is formed by way of example from an elongated cylindrical housing 13 having a chromatographic filter material 14 such as a multitude of cross-linked co-polymer resin beads retained therein by a screen 15 at each end of the column of resin beads. The screen 15 may be a micron mesh stainless steel screen for retaining the resin beads. Such a chromatographic column is described in my US. patent application Ser. No. 530,051, entitled Microsphere Filter and Method of Filtration filed on Feb. 25, 1966. The invention is applicable, however, to other types of chromatographic columns.

Slidably mounted through channels 16 and 17 formed through opposite sides of the cylinder 10 respectively, is a piston 18, having O- ring seals 20 and 21 for engaging the channels 16 and 17, respectively, of the cylinder 10. The O-ring seals are adapted to slide within the channels while maintaining a sealing relationship between the piston 18 and cylinder 10. The portion of piston 18 within the pressure chamber 22 defined by the cylinder housing 10 is provided with differential diameters D and D The upper portion of piston 18 within the pressure chamber 22 of diameter D is of narrower diameter than the lower portion of the piston within chamber 22 having a diameter of D so that a net downward force is exerted on the piston 18 when fluid under pressure is admitted into the pressure chamber 22 through an inlet 23 formed in the cylinder for receiving fluid from a pump. An outlet 24 is also provided through the cylinder wall from the pressure chamber 22 for ejecting the fluid under pressure.

The piston 18 is provided with a detachable extension 26 which serves as the head spacer for the chromatographic column. The head spacer 26 may be attached to the piston 18 by complementary threaded screws 27 provided on an extension and a recess in the head spacer 26 and piston 18, respectively. An O-ring seal 28 is provided in the recess to provide a fluid seal between the threaded portions for reasons hereinafter described. The extension 26 which for-ms the head space reducer is formed of dimensions complementary to and slightly smaller than the head space 30 formed in the chromatographic column over the filter material 14. The head spacer 26 is provided with an O-ring seal 31 which slidably engages the inner wall of the housing 13 for the chromatographic column in sealing relationship. The head spacer 26 which is attached to the piston 18 may be any of a variety of sizes chosen to displace the head space in the chromatographic column being used.

An axial passageway 34 is provided down the center of piston 18 and the attached head spacer 26 including an inlet 35 in the piston for receiving a sample and buffer solution and an outlet 36 in the head spacer for depositing the sample and buffer solution conducted down passageway 34 onto the top of the bed of resin beads 14 or other chromatographic filter material.

In operating the automatic head space reducer, the head spacer 26 is screwed into the piston 18 and positioned within the head space 30 of a chromatographic colum. The inlet 35 to passageway 34 within the piston 18 is opened to permit a buffer solution to fill the open head space between the top of the column of resin beads 14 and the head spacer 26. The mountings 11 in cylinder 10' are then positioned down on the piston against the bottom stops 36- formed on the piston and secured with respect to the chromatographic column so that the cylinder is as far down on the piston as possible. A buffer solution under pressure is then admitted through the inlet 23 into pressure chamber 22 of the cylinder 10 so that a downward force which is a function of the fluid pressure and the differential area between the portions of the piston of diameter D and D is applied to the piston. The differential area on piston 18 within the pressure chamber 22 is chosen so that the resultant force slightly exceeds the upward force on the piston from the back pressure from the chromatographic column and the frictional force of the O- ring seals 20, 21 and 31 so that the piston and head spacer slide downward to rest on the screen 15 and filter material 14 with a predetermined minimal force. A retainer ring 19* is provided around the top of the piston to prevent the piston from travelling downward further than a predetermined distance.

Referring to FIG. 4, by way of example, butter solution is applied to the fluid line at a predetermined pressure by the pump 40. A filter 41 in the fluid line filters the buffer solution which then enters the pressure chamber 42 within the cylinder. From the pressure chamber 42, the fluid is released into a sample injector 43 which provides a known resistance in the fluid line injecting the sample into the buffer solution. The injected sample and buffer solution are then conducted through the center of the piston and head spacer 44 to the chromatographic column and out the output end of the fluid line.

Thus, if a pump pressure of P is provided the upward force F due to back pressure from the chromatographic column 45 on the piston and head spacer 44 is given by the following equation where D is the diameter of the head spacer 26 at the end adjacent the chromatographic filter material.

The downward force F on the piston and head spacer 44 from pressure chamber 42 is given by the following equation where D and D are the differential diameters, respectively, of the portion of the piston within the pressure chamber 22 and R is the resistive pressure provided in the fluid line by the sample injector 43 which resistive pressure may be controlled to equal zero.

The downward force F on the piston and head spacer 44 must be chosen to slightly exceed the upward force F and the frictional resistance of the O-ring seals on the piston and head spacer so that the head spacer comes to rest on the column of resin beads or other filter material with a predetermined minimal force.

The materials of the automatic head space reducer must be chosen so that the cylinder and piston and head spacer and column have compatible coefficients of thermal expansion.

In the embodiment of the invention illustrated in FIG. 2 an automatic head spacer reducer is shown wherein frictional forces from the O-ring. seals are minimized by reducing the area of O-ring seal contact between the piston and cylinder and the head spacer and chromatographic column. As shown in FIG. 2, the piston 50 is of reduced diameter and extends through a single channel 51 in a cylinder 52 which defines a pressure chamber 53. The piston 50 terminates within the pressure chamber with a top pressure surface 54 including a depending diaphragm 55 extending between the perimeter of the pressure surface 54 and the bottom of the cylinder 52. The piston is adapted to slide within the channel 51 in sealing engagement with O-ring seal 56 upon application of pressure to the top pressure surface 54. Inlet 57 and outlet 58 are provided in the cylinder communicating with the pressure chamber 53 for receiving and discharging a fluid under pressure for transmitting pressure to the top pressure surface 54. Attached to the end of the piston 50 outside the pressure chamber is a connector 60 which is in turn connected to a head spacer 61 which may be of variable size for displacement of head space in the chromatographic column with which it is to be used. The piston 50, connector 60 and head spacer 61 may be connected by suitable threaded extensions and recesses. The connector 60 includes an inlet 62 for receiving a buffer solution and sample for delivery through the axial channel 63 formed down the center of the connector 60 and head spacer 61. An O-ring seal 64 is provided in the threaded recess 65 in the connector 60 for sealing engagement between the connector and head spacer 61 to provide a continuous channel 63. The buffer solution and sample may then be delivered through the channel 63 to the head space 66 over the column of chromatographic material 67 in the chromatographic column 68 which the head spacer 61 is seated. The head spacer includes an O-ring 70 to provide sealing engagement between the head spacer 61 and the wall of the chromatographic column 68.

The automatic head space reducer illustrated in FIG. 2 may be incorporated in a fluid circuit such as illustrated in FIG. 4. The area of top pressure surface 54 is chosen to provide a downward force on the piston from pressure in the fluid line, which exceeds the upward force from the back pressure on the head spacer 61 from the chromatographic column and the' frictional force of the O- ring seals 51 and 70 so that the head spacer comes to rest upon the top of the column of chromatographic filter material with a predetermined minimal force. Because the contact area between the O-ring seals and sliding surfaces has been reduced, a smaller differential force may be used and there is less likelihood of surging. The automatic head space reducer is operated in the manner heretofore described with the cylinder 52 mounted over and in alignment with the chromatographic column 68.

In the head space reducer illustrated in FIG. 3, the walls 80 of a chromatographic column 81 provide the side walls for the pressure chamber 82 in which fluid under pressure is received. The top of the pressure chamher comprises a stopper 83 placed in the head of the chromatographic column and in sealing engagement therewith by means of O-ring seal 84. The stopper 83 and walls thus provide a cylinder which defines the pressure chamber 82. The stopper 83 includes an inlet 85 for receiving fluid under pressure and an outlet 86 for rejecting the fluid and is mounted stationary relative to the chromatographic columns by means of a mounting frame 87. A piston 88 positioned in the head space of the chromatographic column over the chromatographic filter material 90 is provided with a narrow shaft 91 slidably extending through a channel 92 provided centrally through the stopper 83. O- ring seals 93 and 94 provide a sealing engagement between the stopper 83 and shaft 91. The piston 88 and shaft 91 include an axial passageway 95 longitudinal down the center of the shaft and piston and an inlet 96 for receiving buffer solution and sample fluid and delivering the butter and sample through the passageway 95 and out the opposite end 97 of the channel 95 to the column of chromatographic filter material 90.

The piston 88 is provided with tapered surfaces 98 and 100, tapering inwardly from larger diameters at the ends to a central recess of smaller diameter where a large O-ring seal 101 is seated. With the O-ring 101 centered between the tapers 98 and 100, a force downward on the head surface 102 of piston 88 and O-ring 101 slightly greater than the upward force due to back pressure from the chromatographic column on the bottom surface 103 will push the piston downward against the column of chromatographic material to rest thereon with predetermined minimal force. The O-ring seal 101 will slide easily along the wall 80 of the chromatographic column 81 under the slight difierential pressure as long as it is seated in the recess centered between the tapered sides 98 and of the piston 88. However, under violent pressure changes and pressure surge from a displacement pump in the fluid line, the O-ring seal 101 will lodge between the wall 80 of the chromatographic column and either of the tapered surfaces 98 and 100 to restrict movement of the piston 88 and thereby act as a shock absorber to absorb surging pressures in the fluid line from the displacement pump or other sources. The hammer effect on the resin surface due to pump variation will therefore be minimized.

The automatic head space reducer illustrated in FIG. 3 will tolerate a greater differential coefiicient thermal expansion between the head spacer piston 88 and the chromatographic column than in the previous embodiments because the O-ring seal is larger and is not held in a fixed slot. On the other hand, the larger O-ring seal 101 does not increase the friction because under the normal ditferential pressure from pressure chamber 82 the O-ring seal 101 is centered between the tapered surfaces 98 and 100 so that it slides easily within the walls of the chromatographic column. Thus, when pressure is applied to the fluid line by a buffer solution pump, the head spacer piston 88 will come to rest on the column of chromatographic filter material with a predetermined minimal force and follow the column of filter material downwardly upon compaction of the filter material under pressure and upwardly upon expansion of the filter material thereby continually eliminating the head space and controlling the pressure on the filter material.

In operation of each of the head space reducers illustrated in FIGURES 1 through 3, a priming butter solution is first fed down the central axial channel within the piston to fill the head space above the chromatographic column so that the fluid line is primed before the buffer solution pump is turned on. With the pump turned off, difierential forces on the piston cause the piston to slide downward following the compacting column of filter material, continuously eliminating the head space.

FIGURES 5 and 6 illustrate an automatic head space reducer in which the need for a pressure chamber upstream from and separate from the sample injector input to the chromatographic column is eliminated. In the embodiment of the invention illustrated in FIGURES and 6, fluid under pressure received in a buffer solution and sample inlet itself provides the pressure for driving a piston to displace the head space within a chromatographic column. As shown in those figures, a stopper or cylinder 110 is provided over a chromatographic column on a mounting frame 111. The outside of the cylinder 110 is in sealing engagement with the Wall 112 of the chromatographic column 113 by means of O-ring seal 114. The cylinder 110 includes a longitudinal recess 115 extending axially from the bottom end within the chromatographic column and further includes a tubular passageway 116 extending axially within the longitudinal recess 115 and out the top of the cylinder to an inlet opening 117 for receiving buffer solution and injected sample fluid under pressure. The piston comprises a piston head spacer 118 adapted to fit within the head space of a chromatographic column and a tubular channel 120 extending through the piston head 118 and upwardly therefrom. The tubular channel 120 is adapted to axially enclose the tubular passageway 116 in sliding relationship. The tubular channel extends within the longitudinal recess 115 in the cylinder 110 and at its outer surface engages an O-ring seal 121 in the longitudinal recess 115 to provide sealing engagement. The longitudinal recess 115 thereby defines a pressure chamber for exerting a downward force on the piston 118 via a tubular channel 120. The tubular channel 120 terminates at the bottomend of the piston 118 adjacent the chromatographic column in a ball valve comprised of a ball 122 of metal or other suitable material seated within a complementary orifice in the base of the piston. The ball 122 is retained under pressure against the channel 120 by a membrane 123 held against the bottom of the piston head 118 with a predetermined force. The membrane 123 may form the bottom of a cap 124 to screw over the bottom of piston head 118 along threads provided thereon. The membrane 123 may be a plastic membrane or other suitable membrane having holes therein for distributing fluid along the top of the column of chromatographic filter material. The membrane 123 retains the ball 122 in position locking the channel 120 with a predetermined force of, for example, a half pound.

In operating the automatic head space reducer shown in FIGURES 4 through 6, the head space over a chromatographic column is first filled with a buffer solution andthe piston 118 is positioned in the head space with the cylinder 110 mounted over the chromatographic column. A buffer solution under pressure is received in the inlet 117 through a fluid line 126 and fed to the tubular channel 120 of the piston 118.

Buffer solution under pressure is received in the inlet 117 from fluid line 126 and is fed through the tubular passageway 116 to the tubular channel 120 so that a net downward force is applied to the piston 118. The tubular channel 120 slides downward within the longitudinal depression 115 and the piston head 118 displaces buffer solution in the head space in the column of chromatographic filter material, to come to rest on the top of the column of chromatographic filter material with a predetermined minimal force. The pressure of the piston on the chromatographic filter material is determined by the release pressure of the ball valve comprising the ball 122 and membrane 123. The valve may be adjusted to provide release of buffer solution and sample from the tubular channel 120 through the membrane 123 upon creation of a differential force downward in the tubular channel 120 of, for example, a half pound. Thus, the piston 118 will come to rest on the column of chromatographic filter material with a pressure of a half pound, the excess pressure producing release of butter solution and injected sample under pressure through the tubular channel 120. The piston 118 which displaces the head space at the top of the chromatographic column is thereby driven directly by the fluid pressure from the buffer solution and injected sample inlet 117.

What I claim is:

1. An automatic head space reducer for a chromatographic column having'an elongated housing, chromatographic filter material retained within the housing, and head space within the housing over the filter material and a fluid line for delivering buffer solution and samples to the column comprising:

displacement means positioned in the head space of the chromatographic column and slidably engaging the housing of the chromatographic column, said displacement means slidable in response to downward forces produced by pressure in the fluid line and upward forces produced by back pressure transmitted from the chromatographic column, said displacement means adapted to rest, in response to pressure from the fluid line, on the chromatographic filter material with a downward force in excess of upward force, due to back pressure, by a predetermined amount, said displacement means having a channel for delivering fluid from the fluid line to the chromatographic filter material,

and means transmitting downward pressure from the fluid line to said displacement means.

2. An automatic head space reducer for a chromatographic column having an elongated housing, chromatographic filter material retained within the housing, head space within the housing over the filter material, and a fluid line for delivering buffer solution and samples to the column comprising:

cylinder means positioned over and fixed relative to the chromatographic column, said cylinder means having an inlet adapted to receive fluid under pressure from the fluid line and an outlet adapted to expel fluid received under pressure;

and piston means positioned within the head space of the chromatographic column and slidably engaging said cylinder means, said piston being slidable within the head space of the chromatographic column in response to fluid pressure transmitted through the fluid line and cylinder means, said piston means adapted to deliver fluid from the cylinder means to the filter material through a channel formed through the piston, said piston means adapted to rest on the column of filter material with a downward force, due to pressure in the fluid line, in excess of the upward force, due to pressure on the piston, by a predetermined amount.

3. An automatic head space reducer as set forth in claim 2 wherein the outlet from said cylinder means comprises an elongated tubular passageway and wherein said piston comprises an elongated tubular inlet axially enclosing and slidable in sealing engagement, relatlve to the elongated tubular passageway from the cylinder means, and a piston head connected to the tubular lnlet including an internal channel and an outlet and valve means for releasing through said outlet to the chromatographic column, fluid received from said cylinder means having a pressure in excess of a predetermined amount.

4. An automatic head space reducer as set forth in claim 3 wherein said valve comprises a ball seated in the outlet from said piston and a membrane retained under pressure against said ball and piston.

5. An automatic head space reducer as set forth in claim 2 wherein part of said cylinder means comprises the housing of the chromatographic column, and wherein a stopper is provided at the top of the chromatographlc column housing having an inlet and outlet for receiving and delivering fluid under pressure from the fluid line, and a channel for receiving said piston means in slidable relationship.

6. An automatic head space reducer as set forth in claim 5 wherein the piston means includes a piston head and an O-ring seal for sealing engagement between the piston head and housing of the chromatographic column, said piston head having sides tapering from maximum widths at each end to a minimum width in the middle, said O-ring seal positioned on the piston substantially at the point of minimum width, said piston means also including a passageway therethrough for receiving and delivering a sample to the chromatographic cloumn.

7. An automatic head space reducer for a chromato graphic column having an elongated housing, chromatographic filter material retained within the housing, head space within the housing over the filter material and a fluid line for delivering buffer solution and samples to the column comprising:

a pressure chamber having an inlet for receiving fluid under pressure from the fluidline and an outlet for releasing received fluid into the fluid line, said pressure chamber being positioned over and fixed relative to a chromatographic column, said pressure chamber having at least one channel for receiving a piston;

elongated piston means positioned in the head space of the chromatographic column and communicating with said pressure chamber through the receiving channel, said piston means slidable within the head space of the chromatographic column in response to fluid pressure in said pressure chamber, said piston means including a longitudinal passageway therethrough for receiving and delivering a sample and fluid from the fluid line to the chromatographic col umn, said piston adapted to rest with a predetermined minimal differential force on the chromatographic filter material in response to fluid pressure in the fluid line.

8. An automatic head space reducer as set forth in claim 7 wherein said piston means includes an O-ring seal for sealing engagement with the inside of said chromatographic column housing.

9. An automatic head space reducer as set forth in claim 7 wherein said piston means includes a plurality of attachable sections for adjusting the length of said piston.

10. An automatic head space reducer as set forth in claim 7 wherein said pressure chamber comprises one channel for receiving a piston and said piston comprises a relatively narrow section extending through said channel, a relatively wide pressure surface within said pressure chamber and a diaphram depending from the perimeter of said pressure surface to a wall of said pressure chamber.

11. An automatic head space reducer as set forth in claim 7 wherein said pressure chamber includes two channels in alignment on opposite sides for. receiving a piston therethrough and said piston extends through the pressure chamber, said piston comprising sections of differential diameter within the pressure chamber adapted to provide a force downward on the piston in excess of force upward, due to back pressure on the piston, by a predetermined amount.

12. An automatic head space reducer as set forth in claim 7 wherein the side walls of said pressure chamber comprise the housing of the chromatographic column and said piston comprises a piston head slidable within the head space of the chromatographic column, said piston head including an O-ring seal for sealing engagement with the housing of the chromatographic column.

13. An automatic head space reducer system for chromatographic columns comprising:

a fluid line including pump means for delivering fluid under pressure in said line;

a pressure chamber having an inlet for receiving fluid under pressure from said line and an outlet for releasing received fluid and at least one channel for receiving a piston means;

a chromatographic column comprising an elongated housing, chromatographic filter material retained in said housing, and head space within said housing over the filter material;

elongated piston means positioned in the head space of the chromatographic column communicating with said pressure chamber through the receiving channel, said piston means slidable within the head space of the chromatographic colunm relative to the pressure chamber in response to fluid pressure in the pressure chamber, said piston means including a longitudinal passageway therethrough having an inlet and an outlet;

sample injector means having an inlet and an outlet;

said fluid line including means connecting the output from said pressure chamber to the inlet to said sample injector, and the outlet from said sample injector to the inlet to said piston means whereby a sample may be delivered through the outlet from said piston means to the chromatographic column,

said piston means adapted to rest on the chromatographic filter material with a predetermined differential force downward in response to fluid pressure in the fluid line and pressure chamber.

14 An automatic head space reducer for a chromatographic column having an elongated housing, chromatographic filter material retained within the housing, and head space within the housing over the filter material comprising:

cylinder means positioned over and fixed relative to the chromatographic column, said cylinder means comprising an elongated tubular passageway having an inlet adapted to receive fluid under pressure and an outlet adapted to expel fluid under pressure;

and piston means positioned within the head space of the chromatographic column and slidably engaging said cylinder means, the piston means slidable within the head space of the chromatographic column in response to fluid pressure transmitted from said cylinder means, said piston comprising an elongated tubular inlet axially enclosing and slidable relative to the elongated tubular passageway fro-m the cylinder means, and a piston head connected to the tubular inlet, said piston head having an internal channel and an outlet and valve means in said outlet for releasing through said channel and outlet to the chromatographic column, fluid received from said cylinder when a predetermined pressure differential occurs across said valve means.

15. An automatic head space reducer as set forth in claim 14 wherein said valve comprises a ball seated in the outlet from said piston head and a membrane retained under pressure against said ball and piston head.

References Cited UNITED STATES PATENTS 1,453,310 5/1923 Engel 210-350 X JAMES L. DECESARE, Primary Examiner US. Cl. X.R. 55-386; 210-351

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