Sample injection system for liquid chromatography

Abstract

Device (10; 110; 210) for conveying one or more fluids through an outlet line (44), comprising: a housing (20; 120; 220) with a chamber (22; 122; 222) for receiving and / or holding one or more pressurized fluids and an outer surface (24; 124), wherein the housing (20; 120; 220) has a first outlet opening (32; 132; 232), a first inlet opening (30; 230), a second outlet opening (34; 234) and a second inlet opening (36; 236), each opening extending from the chamber (22; 122; 222) to the outer surface (24; 124) to receive conveying means; a first outlet conveying means (42; 142; 242) for connection to an analytical device (65), wherein the first outlet line means (42; 142; 242) is received from the first outlet opening (32; 132; 232) and is in communication with the chamber (22; 122; 222) to guide fluids out of the chamber via the first outlet opening; a first inlet line means (40; 140;240) for connection with a first feed means (70; 170), wherein the first inlet line means (42; 140; 240) is received by the first inlet opening and communicates with the chamber (22; 122; 222) to supply a first fluid into the chamber (22; 122; 222); a second inlet line means (46; 146; 246) for connection with a second feed means (95; 195), wherein the second inlet line means (46; 146; 246) is received by the second inlet opening (36; 136; 236) and communicates with the chamber (22; 122; 222) to supply a second fluid into the chamber; a second outlet line means (44; 144; 244) for connection with a waste container, wherein the second outlet line means (44; 144; 244) is received from the second outlet opening (34; 234) and is in communication with the chamber (22; 122; 222) to guide fluids out of the chamber via the second outlet opening (34; 234);a fitting (241, 243, 245, 247) arranged between each opening (232, 230, 234, 236) and the associated conduit means (242, 240, 246, 244) and comprising a first valve means configured to form an intermediate fluid seal and to retain the conduit means in each opening at a chamber pressure between 15,000 and 120,000 psi; a first outlet conduit valve means (52; 152) arranged in the first outlet conduit means (42; 142; 242) configured to assume a closed position to prevent fluid flow and an open position to allow fluid flow, the valve means being configured to respond to a signal; a first supply means (70; 170) connected to the first inlet conduit means (40; 140;240) connected and configured to assume a supply state to supply the first fluid at a pressure up to a maximum of 120,000 psi and configured to assume a stop state in which the first fluid is not supplied; and a control means (80; 180) for controlling the first outlet line valve means (52; 152) and the first supply means (70; 170), wherein the control means (80; 180) is configured to send a signal (82; 182) to the first outlet line valve means (52; 152) to assume either the open or the closed position, and is configured to send a signal (72; 172) to the first supply means (70; 170) to assume either the supply state or the stop state.;

Description

REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Patent Application No. 60 / 538,683, filed January 23, 2004. The contents of this application are hereby incorporated by reference.FIELD OF THE INVENTIONThe invention relates generally to liquid chromatography and more particularly to operating at high, very high and ultra high pressures for liquid chromatography.BACKGROUND OF THE INVENTIONAs used herein, the term "high pressure liquid chromatography" (HPLC) refers to chromatography performed at pressures up to 4000 psi (1 psi = 6894.76 Pascal). There has been interest in performing liquid chromatography at elevated pressures, such as very high pressure liquid chromatography (VHPLC): 6000-20000 pounds per square inch (psi) and ultra high pressure liquid chromatography (UHPLC): 20000-120000 psi. At pressures greater than 6000 psi, injection valves commonly used in HPLC applications fail due to leakage.When operations are performed online with a liquid chromatography separation column, an apparatus must change between supplying solute and supplying sample to the column. Valves are the major means used to switch between the solute and the sample. The rotary injection valve is a mechanism in which the solute in one valve position is directed to the column and the solute in another position of the valve is directed through a loop containing a sample, thus introducing the sample as a discrete part into the flowing stream. This mechanism works well when the pressure required to make the injection is less than 5000 psi. However, the improved chromatographic resolution of columns packed with smaller diameter particles that require higher pressures to move liquids therethrough directs the industry toward columns where increased pressure is necessary. Stiffening the valves associated with the rotary injection device allows the rotary injection valve to be operated at pressures up to 15000 psi, but excessive wear occurs and the valves have a shorter life.A method of using fluids within capillaries and nanoscale channels (capillaries and channels having an inner diameter of less than 200 microns are referred to as capillaries and nanoscale channels) to act as an on / off valve by freezing and thawing the liquid is known (see, for example, U.S. Patent Nos. 6,159,744 and 5,795,788). It has been found that the flow of liquids can be stopped or diverted to another channel or chamber by merely freezing and thawing the liquid contained within a segment of the nanoscale tube or channel. This flow switching device, referred to as freeze-thaw valve (freeze-thaw valve), does not require moving parts and, most importantly, does not contribute dead volume within the analytical system. Freeze thaw valve systems are basically fluidic on / off valves that operate with the fused silica capillaries commonly used for fluid connections and columns in liquid chromatography where pressures greater than 4000 psi occur. Although the activation times for these valves may be in the sub-second range, they close more slowly (typically 5 seconds) than they re-open. Further information on freeze-thaw valves can be found in PCT application PCT / US03 / 28910 and U.S. Patent 6,557,575, which is hereby expressly incorporated by reference.Another means of feeding a sample to a column is a manual process using a high pressure vessel designed for the loading purpose. One end of an HPLC column is placed in the chamber of this vessel at ambient pressure. The chamber is filled with the sample and then the vessel is brought to a high pressure, forcing a portion of the sample onto the top of the column. The high pressure is maintained over a period of time necessary to load a known amount of sample onto the column. After the loading operation, the pressure is lowered back to ambient pressure and the loaded column is then removed from the loading vessel. The loaded column can be immediately installed or stored manually on a separation device and subjected to separation at a later time. In the separation device, a high pressure pump is directly connected to the column, driving a mobile phase through the column and to a detection device, such as a mass spectrometer. This sequence of operations must be repeated for each sample to be analyzed.US 5672810 A describes a gas chromatograph for analysing a selected target component in a liquid sample also containing a solvent, the gas chromatograph having a sample injection port, a temperature control unit, a split ratio control unit and an evaporation control unit.BIDLINGMEYER, B.A. et al.: Improved chromatographic resolution from pressure-induced changes in liquid - solid distribution ratios. In: Separation Science, Vol. 4, 1969, No. 6, pp. 439-446. - ISSN 0037-2366 reports improved chromatographic resolutions resulting from pressure-induced changes in liquid-solid distribution ratios.MACNAIR, John E.; LEWIS, Kenneth C.; JOGENSON, James W.: Ultrahigh pressure reversed phase liquid chromatography in packed capillary columns. In: Analytical Chemistry, Vol. 69, 1997, No. 6, pp. 983-989. - ISSN 0003-2700 deals with how the application of extremely high pressures in liquid chromatography can improve the efficiency and reduce the analysis time for columns packed with small particles.WU, Naijun; LIPERT, J. Andreas; LEE, Milton, L.: Practical aspects of ultra high pressure capillary liquid chromatography. In: Journal of Chromatography A, Vol. 911, 2001, No. 1, pp. 1-12th - ISSN 0021-9673 reports the investigation of a new pressure compensation injection valve intended for use in the UHPLC.There is a need for an apparatus that can deposit a sample on a column in an automated manner and perform the separation through the column at high pressures.SUMMARY OF THE INVENTIONThe present invention relates to a device for conveying one or more fluids through an outlet conduit having the features of claim 1. the device comprises a housing having a number of openings, conduits in the openings and at least one valve means for controlling the flow in the conduits.The housing includes a chamber for receiving and / or holding one or more fluids under pressure and an exterior. The housing has at least one first outlet opening, a first inlet opening, a second outlet opening and a second inlet opening. Each opening extends from the chamber to the outside. Each opening may receive a conduit.A first output line means is received by the first output opening. The first output conduit means is in communication with the chamber for transporting fluids from the chamber out of the first output port. The first exit line means is used for connection to an analytical device such as an HPLC column. The first inlet port receives a first inlet conduit means. The first inlet conduit means is in communication with the chamber for transporting a first fluid into the chamber. The first inlet conduit means is used for connection to a first delivery device, for example a pump.The second inlet port receives the second inlet conduit means. The second inlet conduit means is in communication with the chamber for transporting a second fluid into the chamber. The second inlet conduit means is used for connection to a second delivery device, such as a reservoir or pump. The second output lead means is received by the second output port. The second output conduit means is in communication with the chamber for transporting fluids from the chamber out of the second output port. The second output conduit means is used for connection to a waste receptacle or recycling means.A first output line valve means is disposed in the first output line means. The valve means is configured to react to a signal. The first output line valve means may be operated with a pressure differential along the valve means of up to 120000 psi. The first output line valve means has a closed position in which fluid is prevented from flowing through the valve means and an open position in which fluid is allowed to flow through the valve means. The first output line valve means is responsive to a signal to assume one of the positions. The chamber is therefore configured for receiving fluid from each of the first inlet conduit means and the second inlet conduit means and for draining fluid through the first outlet conduit means and the second outlet conduit means. Preferably, the first fluid is a solute (solute) while the second fluid is a sample fluid.In one embodiment, the first output line valve means is a freeze thaw valve. The first output line means may be a capillary having a first end in the first output port, a central portion external to the housing, and a second end formed as a liquid chromatography column having an input end and an output end. The first output line valve means may be disposed in the central portion of the first output line means. In another embodiment, the first end of the capillary extends into the chamber, with the tip of the first end of the capillary positioned in the chamber between the second inlet port and the second outlet port.In one embodiment, a second output line valve means is disposed in the second output line means. Preferably, the second inlet conduit valve means is disposed in the second inlet conduit means. A second fluid source may be connected to the second inlet conduit for supplying the second fluid. According to the invention, the device further comprises a first supply means connected to the first inlet conduit means. The first supply means has a supply state in which the first fluid is supplied at a pressure that can be up to a maximum pressure. The first supply means also has a stop state in which the first fluid is not supplied. The first supply means is responsive to a signal to assume one of these states.In one embodiment, the device further comprises control means for controlling each of said at least one valve means by sending a signal to said valve means to assume said open or closed position. Preferably, the control means further controls the first supply means by sending a signal to the supply means to assume the supply state or the stop state.In one embodiment, the control means sends a signal to the first supply means to cause a supply state of the first supply means. The control means further sends one or more signals to the at least one valve means to cause a closed position of all the line valve means to fill and pressurize the chamber. Further, the control means sends a signal to the first supply means to cause a stop state of the first supply means and sends one or more signals to the at least one valve means to cause a closed position of the first output line valve means and an open position of the second inlet line valve means and the second output line means to replace a fluid currently present in the chamber with the second fluid. The apparatus may comprise a fluid monitoring device for monitoring a fluid passing through the second output conduit means. The fluid monitoring device is configured to provide the control means with information about the composition of the fluid that exits the chamber. The control means monitors the fluid at the second outlet line and determines the concentration of the second fluid.Preferably, the control means sends a signal to the first supply means to cause a stop state of the first supply means and sends one or more signals to the at least one valve means to cause a closed position of the first output valve means and the second inlet valve means and an open position of the second output valve means to discharge fluid from the chamber. Further, the control means sends a signal to the first supply means to cause a supply state of the first supply means and sends one or more signals to the at least one valve means to cause a closed position of the second inlet valve means and the second outlet valve means and an open position of the first outlet valve means to convey a fluid under pressure through the first outlet line means.In one embodiment, the chamber is configured to maintain fluids at pressures approximately between atmospheric pressure and maximum pressure. In one embodiment, the maximum pressure may be about 5000 psi. In another embodiment, the maximum pressure may be about 15000 psi. In a third embodiment, the maximum pressure may be about 120000 psi, and more specifically, the pressure in the chamber is between about 30000 and 100000 psi when fluid is conveyed through the first output port.Preferably, the embodiments further comprise a vent opening in the housing extending from the chamber to the exterior for receiving a vent conduit means. The vent conduit means received in the vent opening is in communication with the chamber for transporting fluids from the chamber out of the vent opening. The vent line means may be connected to a vent line valve means. The vent line valve means disposed in the vent line means has an open position in which fluid is allowed to flow through the vent line valve means and a closed position in which fluid is prevented from flowing through the vent line valve means. The vent line valve means is responsive to a signal to assume one of the positions.In one embodiment, the control means sends a supply signal to the first supply means to cause a stop state of the first supply means. The control means further sends one or more signals to the at least one valve means to cause a closed position of the first output line valve means and the second inlet line valve means, and an open position of the second output line valve means and the vent line valve means to drain fluid from the chamber.The device according to the invention further comprises at least one fitting arranged between an opening and the associated conduit means and having a first valve means configured to retain the conduit means in the opening at high pressure. The fitting forms a fluid tight seal between the opening and the associated conduit means and retains the associated conduit means in the opening when the pressure in the chamber is increased. According to the invention, the fitting retains the conduit means in the opening at a chamber pressure between 15000 and 120000 psi.In one embodiment, the chamber includes a cylindrical wall, a first end wall, and a second end wall. In this embodiment, the first inlet opening is positioned in one of the first and second end walls and the first outlet opening is positioned in the other end wall. The second inlet port is positioned through the cylindrical wall proximate the first outlet port and the second outlet port is positioned through the cylindrical wall proximate the first inlet port. If a vent is present, then the vent through the cylindrical wall is positioned approximately diametrically opposite the second inlet port.The housing surrounding the chamber comprises an inert material, which may be a stainless steel, titanium or other metals inert with respect to the fluids used.According to the invention, the invention also relates to a method for injecting one or more fluids into an outlet pipe under high pressure, having the features of claim 26; this method uses a device according to the invention comprising a housing having a number of openings, conduits in the openings and at least one valve means to control the flow in the conduits. The method includes providing a housing having a chamber for receiving and / or holding one or more fluids under pressure and an exterior. The housing has at least one first outlet opening, a first inlet opening, a second outlet opening and a second inlet opening. Each opening extends from the chamber to the outside. Each opening may receive a conduit.A first output line means is received by the first output opening. The first output conduit means is in communication with the chamber for transporting fluids from the chamber out of the first output port. The first exit line means is used for connection to an analytical device such as an HPLC column. Preferably, the first output line means is a capillary. The first inlet port receives a first inlet conduit means. The first inlet conduit means is in communication with the chamber for transporting a first fluid into the chamber. The first inlet conduit means is used for connection to a first delivery means, for example a pump.The second inlet port receives the second inlet conduit means. The second inlet conduit means is in communication with the chamber for transporting a second fluid into the chamber. The second inlet conduit means is used for connection to a second delivery means, such as a reservoir or pump. Preferably, the first fluid is a solute and the second fluid is a sample fluid. The second output lead means is received by the second output port. The second output conduit means is in communication with the chamber for transporting fluids from the chamber out of the second output port. The second output conduit means is used for connection to a waste receptacle or recycling means.At least one valve means is disposed in at least one of the first output conduit means, the second output conduit means and the second inlet conduit means. Each valve means of the at least one valve means is operable with a pressure differential along the valve means of from 15000 to 120000 psi. The valve means has a closed position in which fluid is prevented from flowing through the valve means and an open position in which fluid is allowed to flow through the valve means. The valve means is responsive to a signal to assume one of the positions. Preferably the valve means is a freeze-thaw valve.The method comprises receiving a fluid from the first inlet conduit means and / or the second inlet conduit means and draining fluid through the first outlet conduit means and the second outlet conduit means.Preferably, the method further comprises providing a first delivery means, a source of a second fluid, and a control means. The first supply means is connected to the first inlet conduit means. The first supply means has a supply state in which the first fluid is supplied at a pressure that can be up to the maximum pressure, and a stop state in which the first fluid is not supplied. The first supply means is responsive to a signal to assume one of the states. The first supply means may be a pump. The source of the second fluid is in fluid communication with the second inlet conduit means. The control means is configured to control the at least one valve means by sending a signal to the valve means to assume the open or closed position and to control the first supply means by sending a signal to the first supply means to assume the supply state or the stop state.The method comprises causing the control means to send one or more signals to the valve means and to the first supply means to effect a sequence of positions and conditions to move one or more fluids through the first output line means. To inject an amount of the first fluid into the output conduit means, the control means performs the following operations: a. sending a signal to all the conduit valve means to effect a closed condition to seal the chamber, b. sending a signal to the first supply means to effect a supply condition to provide the first fluid and to increase the pressure of the first fluid to a delivery pressure in the chamber, and c. sending a signal to the first output conduit valve means to effect an open position to inject an amount of the first fluid into the first output conduit means.In order to remove the first fluid from the chamber and to inject the second fluid into the outlet conduit means, the control means starts by reducing the pressure in the chamber. The control means performs the following operations: sending a signal to the first output line valve means to cause a closed position; sending a supply signal to the first supply means to cause a stop state; and sending a signal to the second output line valve means to cause an open position. Then, when the pressure in the chamber reaches the ambient pressure, the control means sends a signal to the second inlet line valve means to cause an open position. The control means enables the second fluid to be conveyed into the chamber until the second fluid has displaced the first fluid. Then, the control means brings the pressure in the chamber to the delivery pressure by sending a signal to the second inlet line valve means and the second outlet line valve means to cause a closed position and sending a signal to the first supply means to cause a supply state. While maintaining the pressure in the chamber at the delivery pressure, the control means sends a signal to the first output line valve means to effect an open position for a predetermined period of time to inject the second fluid onto the first output line means.If the apparatus further comprises a fluid monitoring device which monitors a fluid passing through the second outlet conduit, then the monitoring device provides information to the control means about the composition of the fluid exiting the chamber. Using the fluid monitoring information, the control means saves second fluid by delivering the second fluid into the chamber only until the information from the fluid monitoring device indicates that the second fluid has displaced the first fluid. Thus, second fluid is conserved over another method in which second fluid flows for a time sufficient to ensure that the first fluid has been displaced.In order to reduce the pressure in the chamber and discharge fluid from the chamber, the control means sends a signal to the first output line valve means to effect a closed position and a signal to the second output line valve means to effect an open position. If the device further comprises a vent opening in the housing, vent line means and vent line valve means, then the control means also sends a signal to the vent line valve means to effect an open position. This allows fluid to be discharged from the chamber more quickly. The vent opening extends from the chamber to the exterior to receive the vent conduit means. The vent conduit means is received by the vent opening and is in communication with the chamber for transporting fluids from the chamber out of the vent opening. The vent conduit means is adapted for connection to a waste collection device. The vent line valve means is disposed in the vent line means. The vent line valve means is a valve of the type described above having an open and a closed position. The valve means is responsive to a signal to assume one of the positions.If the device includes a vent opening, vent line means and vent line valve means, then the control means may execute a different sequence to remove first fluid from the housing and inject the second fluid into the outlet line means. The control means starts by reducing the pressure in the housing by performing the following operations: sending a signal to the first output line valve means to cause a closed position, sending a signal to the first supply means to cause a stop state, and sending a signal to the second output line valve means and the vent line valve means to cause an open position. When the pressure in the housing reaches the ambient pressure, the control means sends a signal to the vent line valve means to effect a closed position and to the second inlet line valve means to effect an open position. The control means enables second fluid to be delivered into the chamber until the second fluid has displaced the first fluid. Then, the control means brings the pressure in the chamber to the delivery pressure by sending a signal to the second inlet line valve means and the second outlet line valve means to cause a closed position and sending a signal to the first delivery means to cause a delivery state. Once the pressure in the chamber equals the delivery pressure, the control means sends a signal to the first output line valve means to effect an open position for a certain period of time to inject the second fluid onto the capillary.When the apparatus includes a vent opening, a vent line means and a vent line valve means for reducing the pressure in the housing and releasing the second fluid from the chamber, the control means sends a signal to the first output line valve means to effect a closed position and a signal to the second output line valve means and the vent line valve means to effect an open position.Other systems, methods, features and advantages of the present invention will become apparent to those skilled in the art from the accompanying drawings and the detailed description below. It is intended that all such additional systems, methods, features and advantages form part of this specification, be included within the scope of the present invention, and be protected by the appended claims.DESCRIPTION OF THE DRAWINGSNumerous aspects of the invention may be better understood with reference to the accompanying drawings. The components in the drawings are not necessarily drawn to scale, emphasis however being placed upon clearly illustrating the principles of the present invention. Moreover, in the drawings, the same reference numerals have been used in the different views to identify corresponding parts. FIG. 1 shows an illustration of an embodiment of the device according to the invention. FIG. 2 is an illustration of the apparatus of FIG. 1 illustrating a column connected to the apparatus. Figure 3 is an illustration of the apparatus of Figure 1 showing state and control signal connections. FIG. 4 shows a representation of a further embodiment of the device according to the invention. FIG. 5 shows a perspective view of the device of FIG. 4. FIG. 6A shows a top view of the housing of an embodiment of the device of FIG. 5. FIG. 6B shows a detail of an opening in the housing of FIG. 6A. FIG. 6C shows a detail of a feature of the housing of FIG. 6A. FIG. 7A shows a perspective view of the device of FIG. 5, wherein the housing of FIG. 6A is shown in phantom to show connection details of the embodiment. Figure 7B is an illustration of the interior of the chamber of the device of Figure 7A.DETAILED DESCRIPTIONThe present invention relates to liquid chromatography and, more particularly, to an apparatus for conveying one or more fluids through a high pressure liquid chromatography (HPLC) column. The HPLC column is connected to a line leading out of the apparatus. The device comprises a housing having a plurality of apertures, conduits in the apertures and at least one valve means for controlling flow in the conduits.The apparatus 10, as schematically shown in Figure 1, comprises a housing 20 having a chamber 22 for receiving and / or holding one or more fluids under pressure and an outer surface 24. The housing 20 has at least a first outlet opening 32, a first inlet opening 30, a second outlet opening 34 and a second inlet opening 36. Each opening extends from the chamber 22 to the exterior side 24.A first output line means 42 is received in the first output opening 32. The first output conduit means 42 is in communication with the chamber 22 for conveying fluids from the chamber 22 out of the first output port 32. The first output line means 42 is used for connection to an analytical device such as an HPLC column (not shown). The first inlet port 30 receives the first inlet conduit means 40. The first inlet conduit means 40 is in communication with the chamber 22 for conveying a first fluid into the chamber 22. The first inlet conduit means has an inner diameter between about 120 μm and 180 μm. The first inlet conduit means 40 is used for connection to a first delivery device (not shown), such as a pump.The second inlet port 36 receives the second inlet conduit means 46. The second inlet conduit means 46 is in communication with the chamber 22 for conveying a second fluid into the chamber. The second inlet conduit means 46 is used for connection to a second delivery device (not shown), such as a reservoir or pump. The second output line means 44 is received by the second output opening 34. The second output conduit means 44 is in communication with the chamber 22 for delivering fluids from the chamber 22 out of the second output port 34. The second output conduit means 44 is used for connection to a waste receptacle or recycling means (not shown).The chamber 22 is configured to receive fluid from the first inlet conduit means 40 and the second inlet conduit means 46 and discharge fluid through the first outlet conduit means 42 and the second outlet conduit means 44. Preferably, the first fluid is a solute, while the second fluid is a sample fluid. The chamber 22 is further configured to maintain a fluid under a high, very high, and / or ultra high pressure.At least one valve means is arranged at least in the first outlet line means 42, the second outlet line means 44 and / or the second inlet line means 46. Each valve means is operable at a pressure differential along the valve means of about 15000 to 120000 psi. The valve means has a closed position in which fluid is prevented from flowing through the valve means and an open position in which the fluid is allowed to flow through the valve means. The valve means responds to a signal to assume one of these positions. In a preferred embodiment, the at least one valve means is a freeze-thaw valve. Such valves do not have moving parts and do not contribute dead volume to separation, thereby allowing repeatable operation in an inline environment. The freeze defrost valve means is used to control the charging and pressurizing of the device.As can be seen from Fig. 2, a first output line valve means 52 is arranged in the first output line means 42. As shown, the first output line means 42 is a capillary having a first end 62 through the first output port 32, a central portion 64 external to the housing 20, and a second end 66 formed as a liquid chromatography column 65 having an input end 63 and an output end 67. The first output conduit means 42 and the column 65 have an inner diameter of about 15 μm to 150 μm. The first output line valve means 52 is disposed in the central portion 64 of the first output line means 42. As shown, the first end 62 of the first output conduit means 42 extends into the chamber 22, with the tip 68 of the first end 62 of the first output conduit means 42 positioned in the chamber 22 between the second inlet port 36 and the second outlet port 34. This positioning ensures that when the second fluid flows into the second inlet port 36 and out of the second outlet port 34, the area around the tip 68 of the first output conduit means 42 is flushed and any first fluid in the chamber is not retained near the first output conduit means 42.As can be seen from Fig. 3, a second output line valve means 54 is arranged in the second output line means 44. Preferably, a second inlet line valve means 56 is disposed in the second inlet line means 46. A second fluid source 95 is connected to the second inlet line 46 for supplying the second fluid. The second inlet opening and the second outlet opening accommodate conduit means 46, 44 having an inner diameter of about 15 μm to 50 μm.As shown, the apparatus 10 is connected to a first delivery device 70 via the first inlet conduit means 40. The first supply device 70 has a supply state in which the first fluid is supplied at a pressure that can be up to the maximum pressure. The first supply device 70 also has a stop state in which the first fluid is not supplied. The first feeding device 70 responds to a feeding signal 72 to assume one of these states.As shown, the device 10 also comprises a control means 80 for controlling each valve means by sending a signal 82, 84, 86 to the valve means to assume the open or closed position. As shown, the control means 80 further controls the first feeding device 70 by sending a feeding signal 72 to the feeding device 70 to assume the feeding state or the stopping state.In order to fill and pressurize the chamber 22, the control means 80 sends a supply signal 72 to the first supply device 70 to cause a supply state of the first supply device 70. The control means 80 further sends one or more signals to the at least one valve means to cause a closed position of all the line valve means 52, 54, 56 to fill and pressurize the chamber 22.To replace existing fluid in the chamber 22 with the second fluid, the control means 80 sends a supply signal 72 to the first supply device 70 to cause a stop state of the first supply device 70, and sends one or more signals to the at least one valve means to cause a closed position of the first outlet line valve means 52 and an open position of the second inlet line valve means 56 and the second outlet line means 54 to replace a fluid in the chamber 22 with the second fluid. Preferably, as shown, the apparatus 10 includes a fluid monitoring device 90 for monitoring fluid passing through the second output conduit means 44. The fluid monitoring device is configured to provide the control means 80 with a monitoring signal 92 indicative of the composition of the fluid exiting the chamber 22. Preferably, the control means 80 monitors the fluid at the second output line 44 by means of the monitoring signal 92 and determines the concentration of the second fluid in the outlet fluid.To discharge fluid from the chamber 22, the control means 80 sends a supply signal 72 to the first supply device 70 to cause a stop state of the first supply device 70, and sends one or more signals to the at least one valve means to cause a closed position of the first output valve means 52 and the second inlet valve means 56 and an open position of the second output valve means 54 to discharge fluid from the chamber 22.To convey a pressurized fluid through the first output line means 42, the control means 80 sends a supply signal 72 to the first supply device 70 to cause a supply state of the first supply device 70, and sends one or more signals to the at least one valve means to cause a closed position of the second inlet valve means 56 and the second output valve means 54 and an open position of the first output valve means 52 to convey the pressurized fluid through the first output line means 42.In one embodiment, the first supply device 70 is a pump capable of increasing the internal pressure of the chamber 22 to the maximum pressure. The maximum pressure is preferably between 15000 and 120000 psi. The pump may be a binary pump capable of generating a gradient at the maximum pressure. When the chamber is filled with second fluid at the time the pump is switched to the supply state, the pump increases the pressure and compresses the existing fluid. However, the first fluid does not necessarily have to displace the second fluid unless fluid exits the chamber.In one embodiment, the chamber 22 is configured to maintain fluids at pressures that are approximately between atmospheric pressure and maximum pressure. In one embodiment, the maximum pressure ranges to about 5000 psi. In another embodiment, the maximum pressure extends to about 15000 psi. In a third embodiment, the maximum pressure extends to 120000 psi, and more specifically, the pressure in the chamber is approximately between 30000 and 120000 psi as fluid is conveyed through the first output port 32.The device 110 as shown in FIG. 4 further includes a vent opening 138 in the housing 120 extending from the chamber 122 to the exterior 124 to receive a vent conduit means 148. The vent conduit means 148, which is received by the vent opening 138, is in communication with the chamber 122 to convey fluids from the chamber 122 out of the vent opening 138. A vent conduit means 148 has an inner diameter of about 25 μm to 150 μm. As shown, vent line means 148 includes vent line valve means 158 disposed therein. The vent line valve means 158 disposed in the vent line means 148 has an open position in which fluid is allowed to flow through the vent line valve means 158 and a closed position in which fluid is prevented from flowing through the vent line valve means 158. Vent line valve means 158 responds to a signal 188 to assume one of the positions.Generally, the inner diameters of the second inlet conduit means 146 and the second outlet conduit means 144 are the smallest diameters used in the apparatus 110. The first outlet conduit means 142 has the next larger diameter and the vent conduit means 148 and the first inlet conduit means 140 have the largest diameters of the conduit means used in the apparatus 110. This hierarchy allows maximum control of the amount of second fluid used as well as control of the fluids being conveyed to the column (not shown) while minimizing the amount of time taken to decrease the pressure in chamber 122 between process steps.In order to discharge fluid from the chamber 122, the control means 180 of the device of FIG. 4 sends a supply signal 172 to the first supply device 170 to cause a stop state of the first supply device 170. The control means 180 further sends one or more signals to the at least one valve means to cause a closed position of the first output valve means 152 and the second inlet line valve means 156 and an open position of the second output line valve means 154 and the vent line valve means 158 to drain fluid from the chamber 122. The device 110 dissipates pressure more quickly and releases fluids from the chamber 122 more quickly due to the larger diameter of the vent line means 148 and the ability to simultaneously open both the vent line valve means 158 and the second line outlet valve means 154.In implementing the apparatus, such as apparatus 210 as shown in FIG. 5, embodiments may further include at least one fitting, such as fitting 243, disposed between an opening and the associated conduit means. Such a fitting is described in co-pending application 60 / 410,346, which is hereby incorporated by reference. This fitting 243 is designed to form a fluid-tight seal between the opening and the associated conduit means, in particular when the conduit means is formed from quartz glass. Fused silica is not stable enough to withstand the forces exerted by a conventional high pressure ferrule such as ferrule 241. Fitting 243 is also to retain the associated conduit means in the opening when the pressure in the chamber is increased. Fitting 243 retains the tubing in the opening to a chamber pressure of about 15000-120000 psi. If the conduit means is formed of a metallic material such as stainless steel, titanium or other inert metal, as illustrated by the first inlet means 240, then a simpler high pressure fitting such as fitting 241 may be used. This fitting 241 may be a high pressure ferrule. The housing 220 comprises an inert material, which may be stainless steel, titanium or other metal inert to the fluids used and suitable to withstand the maximum pressure.The housing is arranged around a chamber which is preferably between 1 and 3 cm long and has a diameter of 35 to 60 mm. Such dimensions allow small amounts of sample and small amounts of solute to be used. A chamber used was about 1.7 cm long and had a diameter of about 45 mm. The housing 220 to enclose such a chamber preferably has the following dimensions: between 1 and 2 inches long (1 inch = 2.54 cm), between 0.5 and 1.0 inches wide, and 0.35 to 0.7 inches high. A housing used was 1.5 inches×0.7 inches×0.4 inches.In one embodiment, the housing 220 described above has been fabricated as shown in Figure 6A with an inner cylindrical chamber 222 which has approximately a length of 0.65 inch and a diameter of approximately 0.018 inch and thus has a volume of 0.306 inch. The housing 220 was made of 3 stainless steel. Chamber 222 has a side wall 223 around chamber 222 and open ends. A first inlet port 230 was connected to the first end 225 of the chamber 222. The first inlet port 230 was configured to receive a stainless steel Zetali ferrule fitting sold by Waters Corp. (Milford, Massachusetts). A second exit opening 234 has been provided in the side wall 233 of the chamber 222, with the center of the second exit opening 234 having been located approximately 0.017 inch along the side wall 233 from the first end 235 of the chamber 222. The second exit opening 234 was located very close to the first end 235 of the chamber 222 to minimize any remaining first fluid when the second fluid displaced the first fluid. The second end opening 234 has been configured to receive a #6-40 ferrule as shown in FIG. 6C. The first outlet opening 232 has been provided in the connected state with the open second end 227 of the chamber 222, as shown in detail in FIG. 6B. The second inlet port 236 and the vent port 238 have been provided on both sides of the chamber 222 through the side wall 233 at a distance of about 0.035 inch from the second end 227 of the chamber 222. The first output port 232, the second input port 236, and the vent port 238 have been configured to receive a 6-40 ferrule, as shown in FIG. 6C.In an embodiment shown in Figure 7A, wherein the housing 220 of Figure 6A is shown in phantom to expose the chamber 222, the device is comprised of a housing as shown. The first inlet port 230 is provided with a ferrule 231 for retaining a first inlet conduit means 240 in the port. The first inlet conduit means 240 has an inner diameter, which is the largest diameter of the conduit means, to allow for efficient pressurization and purging of the chamber. The first inlet conduit means 240 has an inner diameter of preferably between 120 and 180 μm and an outer diameter of about 360 μm. In one case, the first inlet conduit means had an inner diameter of 150 μm and an outer diameter of 360 μm. The first inlet conduit means 240 is preferably formed of stainless steel and is retained by a compression fitting 241, such as the Waters Z detail, as used in a Waters Alliance HPLC system sold by Waters Corporation of Milford, Massachusetts. Alternatively, a fused silica capillary of the same size range may be used as the first inlet conduit means 240. The quartz glass conduit means retaining mechanism, as described above, would be a high pressure capillary fitting configured to be connected to the ferrule 231 to retain the quartz glass capillary without damaging the capillary.The first output port 232 is provided with a ferrule 233 to retain a first output conduit means 242 in the port. The first output conduit means 242 has an inner diameter that is no greater than the inner diameter of the first input conduit means 240 and is adapted for the chromatographic process to be performed. Typically, the first output conduit means 242 has an inner diameter of 15 to 150 μm and an outer diameter of about 360 μm. In one case, the first output conduit means 242 had an inner diameter of 70 μm and was packed outside the housing 220 with particles forming a column outside the housing. A high pressure capillary fitting 243 configured to interact with the ferrule 233 retains the fused silica capillary in the first exit port 232 without damaging the capillary.The second inlet port 236 is disposed proximate the first outlet port 232. The second inlet port 236 is provided with a ferrule 237 for retaining the second inlet conduit means 246 in the port. The second inlet conduit means 246 has an inner diameter of 15-50 μm and an outer diameter of about 360 μm. In one case, the inner diameter of the second inlet conduit means 246 was 25 μm. A high pressure capillary fitting 247 configured to interact with the ferrule 237 retains the fused silica capillary in the second inlet port 236 without damaging the capillary.The second output port 234 is disposed near the first input port 230. The second output port 234 is provided with a ferrule 235 for retaining the second output conduit means 244 in the port. The second output line means 244 has an inner diameter of 15-50 μm and an outer diameter of about 360 μm. In one case, the inner diameter of the second output line means 244 was 25 μm. A high pressure capillary fitting 245 configured to interact with the ferrule 235 retains the fused silica capillary in the second exit port 234 without damaging the capillary.If an air opening is present, then the air opening 238 is disposed approximately diametrically opposite the second input opening 236. The vent 238 is provided with a ferrule 239 to retain the vent conduit means 248 within the opening. The vent conduit means 248 has an inner diameter of 25-150 μm and an outer diameter of about 360 μm. In one case, the inner diameter of the vent conduit means 248 was 50 μm. A high pressure capillary fitting 249 configured to interact with the ferrule 239 retains the fused silica capillary in the vent 238 without damaging the capillary. Valve means (not shown) is disposed about the first outlet conduit means, the second inlet conduit means, the second outlet conduit means and the vent conduit means outside the housing.FIG. 7B shows how the amount of second fluid to be conveyed into the first output conduit means 242 is controlled by the placement of the tip 268 of the first output conduit means 242. The first output conduit means 242 is inserted into the chamber 222 through the first output port 232. The tip 268 of the first output conduit means 242 is disposed between the second inlet port 236 and the second outlet port 234 at a distance 'd' from the end wall 225. When the chamber 222 is filled with second fluid by introducing second fluid through the second inlet conduit means 246 and discharging it from the second outlet conduit means 244, the volume between the tip 268 of the first outlet conduit means 242 and the end wall 225 is filled with the second fluid. This volume of the second fluid is determined by: where "r" is the radius of the chamber. After chamber 222 is sealed, this volume of second fluid remains between tip 268 and the end of chamber 225. When the pump connected to the first inlet conduit means 240 is switched to a delivery state, the pump attempts to inject first fluid into the chamber 222. However, because the chamber 222 is filled with the second fluid, the pump merely increases the pressure on the second fluid. If the pressure is sufficiently high, then the first output line valve means (not shown) is opened and a quantity of second fluid is conveyed into the first output line means 242 while the same quantity of first fluid displaces the second fluid. This amount of first fluid is contained in the end of chamber 225. The device 210 can only deliver second fluid into the end 268 of the first output conduit means 242 until all of the second fluid has been moved in the volume determined by the equation above. Subsequently, the first fluid provided by the first supply device is conveyed to the conduit means. Although the volume of fluid determined above is an upper limit on the amount of second fluid that can be injected, the time period over which the first output valve means is opened while the chamber 222 is pressurized will usually determine the amount of second fluid injected.In an embodiment where the connections of FIG. 4 have been used with the apparatus of FIGS. 6 and 7, the first inlet line 240, a steel line having an inner diameter of 150 μm and an outer diameter of 1 / 16", has been connected by means of a metallic ferrule fitting (Waters Corporation, Z-Detail Ferrule) between the first inlet port 230 and an ultra high pressure binary gradient pump that has operated as the first feed device 170. The first output line 242, a fused silica capillary having an inner diameter of 75 μm and an outer diameter of 360 μm, was held in the first output port 232 by a high pressure capillary fitting that mates with the 6-40 ferrule port. The second inlet and outlet conduits 246, 244, capillary having an inner diameter of 25 μm and an outer diameter of 360 μm, have been retained in the second inlet and second outlet ports 236, 234, respectively, by means of a high pressure capillary fitting that mates with the 6-40 ferrule port. Vent line 248, a capillary having an inner diameter of 25 μm and an outer diameter of 360 μm, was held in vent 238 by a high pressure capillary fitting that mated with the 6-40 ferrule port. The first outlet conduit 242 was passed through the high pressure fitting until it was positioned with the end of the conduit about 500 μm away from the first end 235 of the chamber 222.The device with the lead held in position has been mounted in a housing. Freeze defrost valve assemblies have been positioned about the first output line 242, the second input line 246, and the second output line 244 and the vent line 248, respectively. In one embodiment, a freeze-thaw valve has been positioned about both the second inlet line 246 and the second outlet line 244, thereby simultaneously controlling flow in these lines. In this embodiment, the second inlet line 246 and the second outlet line 244 have been used exclusively for filling the chamber with the second fluid. Vent line 248 has been used for depressurizing and discharging pressurized fluid from chamber 222.One method of injecting one or more fluids into an output conduit under high pressure uses a device such as that shown in Figure 3. The device comprises a housing 30 having a number of apertures, conduits in the apertures and at least one valve means for controlling the flow in the conduits as described above. The first output line means 42 in the first output port 32 is used for connection to an analytical device such as an HPLC column. The first output line means 42 may be a capillary. The first inlet conduit means 40 in the first inlet port 30 is in communication with the chamber 22 for conveying a first fluid into the chamber 22. The first inlet conduit means 40 is used for connection to a first supply device 70, such as a pump, which supplies a first fluid, such as a solute.The second inlet conduit means 46 in the second inlet port 36 is in communication with the chamber 22 for transporting a second fluid into the chamber 22, The second inlet conduit means 46 is used for connection to a second delivery device 95, such as a reservoir or pump, which delivers a second fluid, such as a sample. The second output conduit means 44 in the second output port 34 is in communication with the chamber 22 for transporting fluids from the chamber 22, and the second output conduit means 44 is used for connection to a waste receptacle or recycle means.At least one valve means is arranged in the first outlet conduit means 42, the second outlet conduit means 44 and / or the second inlet conduit means 46. Each valve means may be operated with a pressure differential along the valve means of up to 120000 psi. The valve means has a closed position in which fluid is prevented from flowing through the valve means and an open position in which fluid is allowed to flow through the valve means. The valve means responds to a signal to assume one of these positions. Preferably, these valves are freeze-thaw valves. The chamber 22 receives fluid from the first inlet conduit means 40 and the second inlet conduit means 46 and discharges fluid through the first outlet conduit means 42 and the second outlet conduit means 44.In addition to the foregoing apparatus, another method employs a first supply device 70, a source of second fluid 95, and a control means 80. The first supply device 70 has a supply state in which the first fluid is supplied at a pressure that can be up to the maximum pressure, and a stop state in which the first fluid is not supplied. The first feeding device 70 responds to a feeding signal 72 to assume the feeding state or the stopping state. The first supply device 70 may be a pump, a binary pump or a gradient pump. The source of second fluid 95 is in fluid communication with the second inlet conduit means 46. the control means 80 is configured to control each valve means by sending a signal 82, 84, 86 to the valve means 52, 54, 56 to assume an open or closed position. The control means is further configured to control the first feeding device 70 by sending a feeding signal 72 to the first feeding device 70 to assume the feeding state or the stopping state.The method includes the control means 80 causing one or more signals to be sent to the valve means and the first delivery device 70 to effect a sequence of positions and conditions to move one or more fluids through the output conduit means 42, 44. To inject an amount of first fluid into the output conduit means 42, the control means 80 performs the following operations: a. sending a signal to all the conduit valve means to effect a closed condition to seal the chamber 22, b. sending a supply signal 72 to the first supply device 70 to effect a supply condition to provide the first fluid and raise the pressure of the first fluid to a delivery pressure in the chamber 22, and c. sending a signal 82 to the first output conduit valve means 52 to effect an open position to inject an amount of the first fluid into the first output conduit means 42.To remove first fluid from the chamber 22 and inject second fluid into the output conduit means 42, the control means 80 starts by reducing the pressure in the chamber 22. The control means 80 performs the following operations: sending a signal 82 to the first output line valve means 52 to cause a closed position, sending a supply signal 72 to the first supply device 70 to cause a stop state, and sending a signal 84 to the second output line valve means 54 to cause an open position. Then, when the pressure in the chamber 22 reaches the ambient pressure, the control means 80 sends a signal 86 to the second inlet line valve means 56 to cause an open position. The control means 80 allows second fluid to be supplied into the chamber 22 until the second fluid has displaced the first fluid. Then, the control means 80 brings the pressure in the chamber 22 to the delivery pressure by sending a signal 86, 84 to the second inlet line valve means 56 and the second outlet line valve means 54 to cause a closed position and sending a supply signal 72 to the first supply device 70 to cause a supply state. While the pressure in the chamber is maintained at the delivery pressure, the control means 80 sends a signal 82 to the first output line valve means 52 to effect an open position for a predetermined period of time to inject the second fluid onto the first output line means 42.If the apparatus 10 further comprises a fluid monitoring device 90 which monitors a fluid conducted through the second output line means 44, then the fluid monitoring device 90 provides information to the control means 80 about the composition of the fluid exiting the chamber 22 by means of a monitoring signal 92. Using the fluid information in the monitoring signal 92, the control means 80 may save second fluid during the displacement operation by supplying the second fluid into the chamber 22 only until the information of the monitoring signal 92 indicates that the second fluid has displaced the first fluid.To decrease the pressure in chamber 22 and discharge fluid from chamber 22, control means 80 sends a signal 82 to first output line valve means 52 to effect a closed position and sends a signal 84 to second output line valve means 54 to effect an open position. As can be seen from Figure 4, if the device 110 further comprises a vent 138 in the housing 120, vent line means 148 and vent line valve means 158, the control means 180 effects venting by sending a signal 188 to the vent line valve means 158 to effect an open position. This allows fluid to be discharged from the chamber 122 more quickly. The vent 138 extends from the chamber 122 to the exterior 124 to receive the vent conduit means 148. Vent conduit means 148 is received through vent opening 138 and is in communication with chamber 122 for transporting fluids from chamber 122 out of vent opening 138. Vent line valve means 158 is disposed in vent line means 148. The vent line valve means 158 is a valve of the type described above having an open and a closed position. Vent line valve means 158 responds to a signal 188 to assume one of these positions.If the device 110 includes a vent opening 138, a vent conduit means 148, and a vent valve means 158, then the control means 180 removes first fluid from the chamber 122 and injects the second fluid into the output conduit means 142 using a different sequence of operations. The control means 180 reduces the pressure in the chamber 122 by performing the following operations: sending a signal 182 to the first output line valve means 152 to cause a closed position, sending a supply signal 172 to the first supply device 170 to cause a stop condition, and sending a signal 184, 188 to the second output line valve means 154 and the vent line valve means 158 to cause an open position. Alternatively, if vent line means 148 is of a significantly larger diameter than second outlet line means 144, then only vent line valve means 158 may be placed in the open position to reduce pressure. When the pressure in the chamber 122 reaches ambient pressure, the control means 180 sends a signal 188 to the vent line valve means 158 to effect a closed position, and the signals 184, 186 to the second output line valve means 154 and the second inlet line valve means 156 to effect an open position. The control means 180 allows second fluid to be delivered into the chamber 122 until the second fluid has displaced the first fluid. Then, the control means 180 brings the pressure in the chamber 122 to the delivery pressure by sending a signal 186, 184 to the second inlet line valve means 156 and the second outlet line valve means 154 to cause a closed position and sending a supply signal 172 to the first supply device 70 to cause a supply state. Once the pressure in chamber 122 is equal to the delivery pressure, control means 180 sends a signal 182 to first output line valve means 152 to cause an open position for a predetermined period of time to inject the second fluid onto output line means 142.In order to reduce the pressure in chamber 122 and discharge the second fluid from chamber 122, in device 110 comprising a vent opening 138, a vent line means 148 and vent valve means 158, control means 180 sends a signal 182 to first output line valve means 152 to effect a closed position and a signal 184 to second output line valve means 154 and vent line valve means 158 to effect an open position.Those skilled in the art will recognize numerous equivalents to the particular procedures, embodiments, claims and examples described herein. Such equivalents are intended to be within the scope of the invention and to be covered by the appended claims.Example:A method in which features of the present inventive apparatus are embodied will be described below with reference to the following example.A column having an inner diameter of 75 μm and a length of 20 cm and packed with C18 particles having a size of 1 μm (Waters Corp., Milford, Massachusetts) was placed in fluid communication with a capillary having an inner diameter of 75 μm. The capillary has been threaded through the first outlet line freeze valve 252 and then through the first outlet high pressure capillary fitting which has been installed in the first outlet line port. The capillary has been inserted into the opening until the tip of the capillary has been located about 500 μm away from the first end 235 of the chamber 222. The capillary is then secured in position by tightening the high pressure capillary fitting.The column was first flushed with a solution of 95% water and 5% ACN by applying 30 kpsi through the pump via chamber 222 with the first output valve 252 in the open position and all other valves in the closed position over a period of 10 minutes. The first output valve 252 was then moved to a closed position and the vent valve 258 was moved to the open position to reduce the pressure to ambient pressure. The vent valve 258 was then moved to the closed position and the second inlet valve and the second output valve 256, 254 were then moved to the open position. The sample, an 8 μM BSA extract, has flowed from the second inlet conduit means 246 to the second outlet conduit means 244, with the solute being replaced until the output of a monitoring device at the output of the second outlet conduit means 244 has indicated that the chamber 222 has been filled with the sample. The second inlet valve and the second outlet valve 256,254 have been moved to a closed position and the pump has been started to apply a 10 kpsi pressure to the sample in chamber 222. The first output valve 252 has been adjusted to the open position and sample has been injected onto the column at 10 kpsi over a period of 0.7 minutes, thereby applying about 1 fmoles of sample to the column. The first output valve 252 has been moved to the closed position and the vent valve 258 has been moved to the open position to remove the sample from the chamber 222. The vent valve 258 has been moved to the closed position and the pump has also been activated to bring the fluid in the chamber 222 to a pressure of 30 kpsi. The pump then has performed a gradient of 20 minutes (5%A→95%B), A=5% ACN, B=70% ACN, each with 0.1% formic acid. The output of the column was examined using a Waters Micromass Q-TOF mass spectrometer (Waters Corp., Milford, Massachusetts) providing separation with narrow Gaussian shaped peptide peaks indicating that a well characterized sample band had been injected onto the column.The described injection device can be used in an automated manner to sequence samples and then solute through a column without manual intervention. The described device can be used in applications ranging from high pressure injections to ultra high pressure injections. The apparatus has better reproducibility than separations performed in separate loading and separating apparatuses. This improvement can be attributed to better control of the amount of sample injected.Those skilled in the art will recognize other features and advantages of the invention based on the above-described embodiments. Thus, the invention is not limited to what has been particularly shown and described, unless indicated by the appended claims.

Claims

An apparatus (10; 110; 210) for conveying one or more fluids through an output conduit (44), comprising: a housing (20; 120; 220) having a chamber (22; 122; 222) for receiving and / or holding one or more pressurized fluids and an exterior (24; 124), the housing (20; 120; 220) having a first output port (32; 132; 232), a first input port (30; 230), a second output port (34; 234), and a second input port (36; 236), each port extending from the chamber (22; 122; 222) to the exterior (24; 124) to receive conduit means; a first output conduit means (42; 142; 242) for connection to an analytical device (65), the first output conduit means (42; 142; 242); 242) received from the first output port (32; 132; 232) and in communication with the chamber (22; 122; 222) for carrying fluids out of the chamber via the first output port; first inlet conduit means (40; 140; 240) for connection to a first supply means (70; 170), the first inlet conduit means (42; 140; 240) being received from the first inlet port and in communication with the chamber (22; 122; 222) for carrying a first fluid into the chamber (22; 122; 222); second inlet conduit means (46; 146; 246) for connection to a second supply means (95; 195), the second inlet conduit means (46; 146; 246) being received from the second inlet port (36; 136; 236) and in communication with the chamber (22; 122; 236); 222) for carrying a second fluid into the chamber; second output line means (44; 144; 244) for connection to a waste receptacle, the second output line means (44; 144; 244) being received by the second output port (34; 234) and being in communication with the chamber (22; 122; 222) for carrying fluids out of the chamber via the second output port (34; 234); a fitting (241, 243, 245, 247) disposed between each port (232, 230, 234, 236) and the associated line means (242, 240, 246, 244) and having first valve means configured to form an intermediate fluid seal and to maintain the line means in each port at a chamber pressure between 15,000 and 120,000 psi; a first output line valve means (52; 152) disposed in said first output line means (42; 142; 242) and configured to assume a closed position to prevent a flow of fluid and an open position to allow a flow of fluid, said valve means being configured to respond to a signal; a first supply means (70; 170) connected to said first inlet line means (40; 140; 240) and configured to assume a supply state to supply said first fluid under a pressure up to 120,000 psi at maximum and configured to assume a stop state in which said first fluid is not supplied; and a control means (80; 180) for controlling said first output line valve means (52; 152) and said first supply means (70; 170), said control means (80; 180) is configured to send a signal (82; 182) to the first output line valve means (52; 152) to assume either the open or the closed position, and is configured to send a signal (72; 172) to the first supply means (70; 170) to assume either the supply state or the stop state.The apparatus (10; 110; 210) of claim 1, wherein the first output line valve means (52; 152) is a freeze thaw valve.The apparatus (10; 110; 210) of claim 2, wherein the first fluid is a solute.The apparatus (10; 110; 210) of claim 2, wherein the second fluid is a sample fluid.The apparatus (10; 110; 210) of claim 1, wherein the first output line means (42; 142; 242) is a capillary having a first end (62) in the first output port (32; 132; 232), a central portion (64) external to the housing (20; 120; 220), and a second end (66) formed as a liquid chromatography column (65) having an input end (63) and an output end (67), and wherein the first output line valve means is disposed in the central portion (64).The apparatus (10; 110; 210) of claim 5, wherein the first end (62) of the capillary extends into the chamber (22; 122; 222).The device (10; 110; 210) of claim 6, wherein a tip (68) of the first end (62) of the capillary is disposed in the chamber (22; 122; 222) between the second inlet port (36; 236) and the second outlet port (34; 234).The device of claim 5, wherein the capillary has an inner diameter of about 15 μm to 150 μm.The apparatus of claim 1, wherein the apparatus further comprises fluid monitoring means for monitoring a fluid passed through the second output conduit means (44; 144; 244) to provide the control means (80; 180) with information about the composition of the fluid exiting the chamber (22; 122; 222).The apparatus (10; 110; 210) of claim 9, wherein the control means (80; 180) is configured to monitor the fluid at the second output conduit means (44; 144; 244) to determine its concentration.The apparatus of claim 1, wherein the first supply device (70; 170) is a pump adapted to pump fluid to the maximum pressure against an internal pressure of the chamber (22; 122; 222).The device (10; 110; 210) according to claim 11, wherein the pump is a binary pump adapted to generate a gradient at the maximum pressure.The apparatus (10; 110; 210) of claim 1, wherein the apparatus further comprises a second output line valve means (54; 154) disposed in the second output line means (44; 144; 244).The apparatus (10; 110; 210) of claim 1, wherein the apparatus further comprises a second inlet line valve means (56; 156) disposed in the second inlet line means (46; 146; 246).The apparatus (10; 110; 210) of claim 1, wherein the apparatus further comprises a second fluid source connected to the second inlet line valve means for supplying the second fluid.The apparatus (10; 110; 210) of claim 1, the apparatus further comprising: a vent (138; 238) extending within the housing (20; 120; 220) from the chamber (22; 122; 222) to the exterior (24; 124) to receive a vent conduit means; and a vent conduit means (148; 248) received through the vent (138; 238) and in communication with the chamber (22; 122; 222) to direct fluids from the chamber via the vent (138; 238), wherein the vent conduit means (148; 248) is configured for communication with a vent conduit valve means (158).The apparatus (10; 110; 210) of claim 16, wherein the apparatus further comprises a vent line valve means (158) disposed in the vent line means (148; 248) and having an open position in which fluid can flow through the vent line valve means, a closed position in which fluid is prevented from flowing through the vent line valve means (158), and wherein the vent line valve means is responsive to a signal (188) to assume one of these positions.The apparatus (10; 110; 210) of claim 16, wherein the vent conduit means (148; 248) has an inner diameter of about 25 μm to 150 μm.The apparatus (10; 110; 210) of claim 16, wherein the chamber (22; 122; 222) includes a cylindrical wall (223), a first end wall (225), and a second end wall (227).The apparatus of claim 19, wherein the first inlet opening (30; 230) is disposed in one of the first end wall (225) and the second end wall (227), and the first outlet opening (32; 232) is disposed in the other wall.The apparatus (10; 110; 210) of claim 20, wherein the second inlet port (46; 146; 246) is disposed through the cylindrical wall (223) proximate the first outlet port (32; 132; 232), and the second outlet port (34; 234) is disposed through the cylindrical wall proximate the first inlet port (30; 230).The apparatus (10; 110; 210) of claim 21, wherein the vent opening (138; 238) through the cylindrical wall (223) is disposed approximately diametrically opposite the second inlet opening (46; 146; 246).The apparatus (10; 110; 210) of claim 1, wherein the housing (20; 120; 220) comprises an inert material.The apparatus (10; 110; 210) of claim 23, wherein the inert material is stainless steel or titanium.The apparatus (10; 110; 210) of claim 1, wherein the second inlet port (46; 146; 246) and the second outlet port (34; 234) each receive a conduit means having an inner diameter of about 15 μm to 50 μm.A method of injecting one or more fluids into an output line under high pressure using the apparatus of any one of claims 1 to 25, the method comprising: causing the control means (80; 180) to send one or more signals (82; 182, 84; 184, 86; 186) to the valve means (52; 152, 54; 154, 56, 156) and the first supply means (70; 170) to cause a sequence of positions and conditions to pass one or more fluids through the output line means.A method according to claim 26, comprising injecting an amount of the first fluid into the first output line means (42; 142; 242), and a. sending a signal (82; 182, 84; 184, 86; 186) to all of the valve means (52; 152, 54; 154, 56, 156) to effect a closed state to seal the chamber (22; 122; 222); b. sending a supply signal (72; 172) to the first supply means (70; 170) to effect a first supply state to provide the first fluid and to increase a pressure of the first fluid to a delivery pressure in the chamber (22; 122; 222); and c. sending a signal (82; 182) to the first output line valve means (52; 152) to effect an open position for injecting a quantity of the first fluid into the first output conduit means (42; 142; 242).A method according to claim 26, comprising injecting the second fluid into the first output line means (42; 142; 242) and a. sending a signal (82; 182) to the first output line valve means (52; 152) to cause a closed position, sending a supply signal (72; 172) to the first supply means to cause a stop condition, and sending a signal (84; 184) to the second output line valve means (54; 154) to cause an open position to decrease the pressure in the chamber (22; 122; 222); b. sending a signal (86; 186) to the second input line valve means (56; 156) to cause an open position when the pressure in the chamber (22; 122; 222) reaches an ambient pressure; c. Supplying the second fluid into the chamber (22; 122; 222) until the second fluid displaces the first fluid; d. sending a signal (86; 186, 84; 184) to the second inlet line valve means (56; 156) and the second outlet line valve means (54; 154) to cause a closed position and sending a supply signal to the first supply means (70; 170) to cause a supply state and to bring the pressure in the chamber (22; 122; 222) to a delivery pressure; and e. maintaining the delivery pressure in the chamber (22; 122; 222) and sending a signal (82; 182) to the first outlet line valve means (52; 152) to cause an open position for a predetermined period of time to cause the second fluid into the first outlet line means (42; 142; c; 242).The method of claim 28, further comprising monitoring a fluid passing through the second output conduit means (44; 144; 244) to provide information to the control means (80; 180) about the composition of the fluid exiting the chamber (22; 122; 222).The method of claim 28, wherein step c is replaced by the step of: supplying the second fluid into the chamber (22; 122; 222) until the information from the fluid monitoring device (90; 190) indicates that the second fluid has displaced the first fluid.The method of claim 26, further comprising: sending a signal (82; 182) to the first output line valve means (52; 152) to cause a closed position; and sending a signal (84; 184) to the second output line valve means (54; 154) to cause an open position and to reduce the pressure in the chamber and flush the fluid from the chamber (22; 122; 222).The method of claim 26, further comprising: a. sending a signal (82;182,84;184,86;186) to all of the line valve means to effect a closed condition for sealing the chamber (22;122;222); b. sending a supply signal (72;172) to the first supply means (70;170) to effect a supply condition for providing the first fluid and to increase the pressure of the first fluid to a delivery pressure in the housing; and c. sending a signal (82;182) to the first output line valve means (52;152) to effect an open position for injecting an amount of the first fluid into the first output line means (42;142;242.The method of claim 26 further comprising: a. sending a signal (82; 182) to said first output line valve means (52; 152) to cause a closed position, sending a supply signal (72; 172) to said first supply means (70; 170) to cause a stop condition, and sending a signal (84; 184, 188) to said second output line valve means (54; 154) and said vent line valve means (158) to cause an open position to decrease the pressure in said housing; b. sending a signal (188, 186) to said vent line valve means (158) to cause a closed condition and to said second input line valve means (56; 156) to cause an open position when the pressure in said housing reaches an ambient pressure; c. Supplying the second fluid into the chamber (22; 122; 222) until the second fluid displaces the first fluid; d. sending a signal (184, 186) to the second inlet line valve means (54; 154) and the second outlet line valve means (56; 156) to cause a closed position and sending a supply signal (172) to the first supply means (170) to cause a supply state to bring the pressure in the chamber (22; 122; 222) to the delivery pressure; and e. maintaining the chamber pressure at the delivery pressure and sending a signal (82; 182) to the first outlet line valve means (52; 152) to cause an open position for a predetermined period of time to inject the second fluid into the first outlet line means (42; 142; 242).The method of claim 26 further comprising sending a signal (82; 182) to said first output line valve means (52; 152) to effect a closed position, and sending a signal (184, 188) to said second output line valve means (54; 154) and said vent line valve means (158) to effect an open position so as to reduce the pressure in said housing (20; 120; 220) and flush said second fluid from said chamber (22; 122; 222).

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