Title of Invention

AN APPARATUS AND METHOD FOR PERFORMING LIQUID CHROMATOGRAPHY WITH CHROMATOGRAPHY COLUMN

Abstract

High pressure liquid chromatographic apparatus in which a fluid mixture containing at least one solute that is reactive with chromatographically reactive surfaces in the column is loaded into the column, and a number of plugs of different eluant fluids are injected into that column. The injections are made in a manner that minimizes the amount of eluant required. In one embodiment, the injections are made to insure that flow of at least the eluant fluids through the column will occur, preferably with a substantially flat wave front, at speeds corresponding to reduced velocities greater than about 5,000, i.e. at flow rates sufficient to induce turbulent flow in those fluids, thereby minimizing the time required for the entire succession of mixture and eluant fluids to traverse the column. In other embodiments, the injections are made substantially simultaneously at spatially separated points adjacent the entrance to the column or are made in sequence to a single location adjacent the entrance to the column, in either case the fluids then tend to travel through the column at a common group velocity as closely bunched fluid plugs.

Full Text

This invention relates to an apparatus and method of performing liquid chromatography with a chromatography column Background of the Invention The separation process effected through high performance liquid chromatography relies on the fact that a number of component solute molecules in a sample stream of a fluid (known as the mobile phase) flowing through a packed bed of particles (known as the stationary phase) usually in a column, can be efficiently separated from one another. Basically, because each individual sample component has a different affinity for the stationary phase, each such component has a different rate of migration through and a different exit time from the column, effecting separation of the components. The separation efficiency is determined by the amount of spreading of the solute band as it traverses the bed or column. Such separations, particularly in preparative processes, have significant limitations typically occasioned by the batch nature of the processes. Typically, a chromatographic column is first equilibrated by flowing an equilibrating fluid through the column, the latter is then charged or loaded with a fluid mixture containing the solute or solutes sought to be separated, and one or more eluant fluids are flowed sequentially through the column to release bound solute selectively. The eluted solutes are thus temporally separated in the flowstream emerging at the output of the column and the process may be repeated cyclically. At typical flow rates and where the concentration of the desired solutes in the initial fluid mixture are low, the production of significant quantities of the desired solutes can be an unhappily slow and very expensive process with current apparatus and methods that require comparatively long cycle times. Accordingly, the present invention provides a method of performing liquid chromatography with a chromatography column having chromatographically reactive surfaces, comprising the steps of flowing through said column a discrete volume of a liquid mixture containing at least one solute that is reactive with said surfaces, and eluting from said surfaces said solute bound thereto, by flowing eluant fluid through said column, the improvement comprising the step of injecting at least one discrete volume of an eluant fluid into the flowstream in said column such as to maintain minimized spatial step separation between said discrete volumes as the latter traverse said column. Accordingly, the present invention also provides a chromatography apparatus comprising uniform, elongated chromatography column containing chromatographically reactive surfaces, means for injecting into said column a discrete volume of liquid mixture containing at least one solute that is reactive with said surfaces so as to load said column, and means for flowing eluant fluid through the loaded column, the improvement wherein: said means for flowing said eluant fluid comprises means for injecting at least one discrete plug of said eluant fluid into said column adjacent the input of said column so as to maintain minimized spatial step separation between said plug and said discrete volume of liquid mixture as said plug and volume traverse said column. ingle loop injectors with by-pass switching can also be employed. In Figs. 6 and 7, all of the parts of the injectors are identical to those of Figs, 4 and 5 except that a by-pass conduit 92 of minimal storage capacity is used to replace loop coil 82. It will be apparent that yet other known types of injectors can also be advantageously employed in the apparatus of the present invention. Switching operation of a plurality of injectors is preferably under the control of means, such as a known computer or controller to optimize switching time and obtain the desired sequence of plug injections into column 40. Injectors of the type described are preferred in as much as they tend to provide desirably sharp transition boundaries between adjacent plugs of fluid, thus reducing mixing. As shown in Fig. 2, injectors 50, 58 and 66 are coupled to input conduit 74 to column 40 at spaced-apart locations, and it will be understood that in such case, all of those injectors can be operated simultaneously by means such as an appropriately programmed computer, to provide a succession of spatially separated plugs presented to head 76 or may be operated serially to provide even more flexibility in spacing the location of the plugs as the latter transit column 40. Yet, as shown in Fig. 8, wherein like numerals denote like parts, all of the injectors may be coupled directly to port 75 or a single location on conduit 74, and in such case, all of those injectors can be operated serially to provide a succession of spatially separated plugs presented to head 76, In the embodiment depicted in Figure 9, solvent reservoir 130 is in fluid communication with main pump 132, which withdraws equilibrating fluid from reservoir 130 and delivers said fluid, under pressure, to column 148. A quick disconnect junction may optionally be included upstream of main pump 132 in order to allow the end of tubing connecting reservoir 130 and main pump 132 to be sealed to avoid or to minimize leakage and spillage should either occur. Main pump 132 may be any suitable pump means, preferably embodied as a single- headed pump coupled to a pressure gauge 134, pressure sensor or transducer 136, and pulse dampener 138. Pressure at the outlet of main pump 132 is monitored using pressure gauge 134 and pressure transducer 136 in combination with an intemal pressure switch which shuts down the system if the pressure is above a predetermined value. Main pump 132 should be capable of delivering the mobile phase of the chromatography system at a ripple-free, user-selected flow rate. When a single headed pump is used, a pulse dampener 138 is included to minimize ripples or pulsations in flow rate which may occur. Pulse dampener 138 preferably includes a pressurized tetrafluoroethylene (TFE) membrane which flexes during the delivery cycle of main pump 132 and which is restored during the "refresh" cycle of pump operation. The pressure applied to the membrane of pulse dampener 138 is dependent on the pressure at which main pump 132 is operated, being equal to about 80% of the operating pressure of main pump 132. An inline filter 140 is placed downstream of pressure transducer 136 to remove particulate matter from the mobile phase. Preferably a filter capable of removing particles greater than about 0.5μm in diameter is employed, though other filters may also be used in accordance with the invention. Downstream of inline filter 140 is an inlet valve 164 and an outlet valve 166 which selectively establish a first flow path and a second flow path, outlet valve 166 being in fluid communication with column 148. In accordance with the invention, the first flow path is in parallel to the second flow path, and each flow path includes at least one injector valve. Preferably, a plurality of manual injector valves is provided along the first flow path, as is exemplified in Figure 9 by valves 142, 144, and 146. The manual injector valves are preferably provided in series with each other, wherein the outlet of each downstream valve is in fluid communication with the inlet of the adjacent upstream valve (e.g., the outlet of valve 142 is in fluid communication with the inlet of valve 144 and the outlet of valve 144 is in fluid communication with the inlet of valve 146). Sample or eluant may be added to the system through any of manual injector valves 142, 144, and 146, in accordance with the present embodiment. The manual injector valves of the first flow path may be used to determine the flow parameters of a specific chromatographic purification empirically, prior to use of the automatic injection valves 154 for the purification. The volumes accomodated by manual injector valves 142, 144, and 146 may be the same or they may differ in accordance with this embodiment. Each of manual injector valves 142, 144, and 146 is filled via a needle port in the valve handle assembly, which depicts separate LOAD and INJECT positions. Fluid is injected into the manual injector valve while the valve handle is in the LOAD position, using a syringe or other suitable cannula having a square end. The valve handle is rotated to the INJECT position to inject the fluid from the valve into the flow path to column 148. Preferably, a plurality of automated injector valves 154 is provided in series along the second flow path between 164 and 166, as is depicted in Figure 9. Sample or eluant fluid is injected from reservoirs 158 into the second flow path using a pump 156 in fluid communication with each automated injector valve 154, as exemplified in Figure 9. Each pump 156 is under the control of a suitable means, such as a computer program, which determines the time of each injection and the flow rate of fluid pumped. A flowmeter may optionally be included between each pump 156 and the respective injector valve 154 to monitor fluid flow into each automated injector valve. The column 148 employed in this embodiment is as described above in relation to the column depicted in Figure 3. A detector 150 is included downstream of column 148 to detect species that elute from column 148. A flowmeter 152 may optionally be included downstream of detector 150, to monitor flow of the mobile phase through the system. Similarly, valves 160 (which are preferably solenoid valves) are included downstream of detector 150 to divert the eluant stream to a collection vessel. A valve 162 is also included downstream of detector 150 to divert the eluant flow into a waste vessel. While the apparatus of the present invention has been described in terms of a chromatographic column of packed particles, as described in the aforementioned U.S. Patent Application Serial No. 08/552193, the columns useful in the present invention can also be in the alternative form of a capillary tube defining a hollow, elongated channel of substantially uniform internal diameter, the channel being provided with a chromatographically active interior surface. The tube is formed such that turbulent flow will be induced in fluid pumped through the interior at a velocity sufficient to create centrifugal forces in the fluid. It has been found with apparatus of the present invention that a complete cycle involving 100 mL sample plugs, using two different 5 mL eluant can be run within about 15 seconds, providing an extremely high throughput. With the WE CLAIM: 1. A method of performing liquid chromatography with a chromatography column having chromatographically reactive surfaces, comprising the steps of flowing through said column a discrete volume of a liquid mixture containing at least one solute that is reactive with said surfaces, and eluting from said surfaces said solute bound thereto, by flowing eluant fluid through said column, the improvement comprising the step of injecting at least one discrete volume of an eluant fluid into the flowstream in said column such as to maintain minimized spatial step separation between said discrete volumes as the latter traverse said column. 2. The method of performing liquid chromatography as claimed in claim 1 wherein at least said volume of eluant fluid traverses said column at a reduced velocity greater than about 5000. 3. The method of performing liquid chromatography as claimed in claim 1 wherein all of said predetermined discrete volumes are injected substantially simultaneously at positions spatially separated along an extension of the axis of elongation of said column so that said volumes traverse said column as closely bunched but separated fluid plugs. 4. The method of performing liquid chromatography as claimed in claim 1 wherein said discrete volumes are injected sequentially. 5. The method of performing liquid chromatography as claimed in claim 4 wherein said sequential injections are effected at positions spatially separated along an extension of the axis of elongation of said column so that said volumes traverse said column as closely bunched but separated fluid plugs. 6. The method of performing liquid chromatography as claimed in claim 4 wherein said sequential injections are effected at one position located adjacent the input of said column. 7. The method of performing liquid chromatography as claimed in claim 3 comprising the steps of cyclically repeating the injections of said discrete volumes and flowing a predetermined volume of equilibrating fluid through said column between cyclic repetitions of said injections. 8. A chromatography apparatus comprising uniform, elongated chromatography column containing chromatographically reactive surfaces, means for injecting into said column a discrete volume of liquid mixture containing at least one solute that is reactive with said surfaces so as to load said column, and means for flowing eluant fluid through the loaded column, the improvement wherein: said means for flowing said eluant fluid comprises means for injecting at least one discrete plug of said eluant fluid into said column adjacent the input of said column so as to maintain minimized spatial step separation between said plug and said discrete volume of liquid mixture as said plug and volume traverse said column. 9 The chromatography apparatus as claimed in claim 8 wherein at said column and said means for flowing are configured such that the flow of said volume of eluant fluid traverses said column at a reduced velocity greater than about 5000. 10. The chromatography apparatus as claimed in claim 9 wherein said means for injecting provides said plug traversing said column with a substantially plane front extending substantially perpendicularly to the axis of elongation of said column. 11. The chromatography apparatus as claimed in claim 9 wherein said means for injecting provides said plug traversing said column with a substantially plane end extending substantially perpendicularly to the axis of elongation of said column. 12. The chromatography apparatus as claimed in claim 8 wherein said means for injecting comprises a plurality of individual injectors for injecting respective plugs of said eluant fluid and said liquid mixture. 13. The chromatography apparatus as claimed in claim 12 wherein said injectors are positioned for injecting a plurality of different plugs at respective spatially separated positions along an extension of the axis of elongation of said column so that said plugs traverse said column as closely bunched fluid plugs. 14. The chromatography apparatus as claimed in claim 13 having means for controlling said injectors so that the latter operate for injecting said respective plugs substantially simultaneously. 15. The chromatography apparatus as claimed in claim 12 having means for controlling said injectors so that the latter operate for injecting said respective plugs sequentially. 16. The chromatography apparatus as claimed in claim 15 wherein said injectors are positioned for injecting plurality of different plugs as sequential injections effected at one position located adjacent the input of said column. 17. The chromatography apparatus as claimed in claim 15 wherein said injectors are positioned for injecting a plurality of different plugs at respective spatially separated positions along an extension of the axis of elongation of said column so that said plugs traverse said column as closely bunched fluid plugs. 18. The chromatography apparatus as claimed in claim 12 having means for cyclically repeating the injections of said plugs of eluant fluid and mixture. 19. The chromatography apparatus as claimed in claim 18 having means for effecting a flow of a predetermined volume of equilibrating fluid through said column between cyclic repetitions of said injections. 20. The chromatography apparatus as claimed in claim 18 wherein said means for injecting comprises at least first and second injectors connected for injecting respective said plugs at respective first and second spatially separated positions along an extension of the axis of elongation of said column, and at least third and fourth injectors connected for injecting respective said plugs at third and fourth spatially separated positions, said apparatus having means for controlling the injections by said injectors for cyclically alternating injections of said respective plugs by said first and second injectors at said first and second positions^ with injections of said respective plugs by said third and fourth injectors at said third and fourth positions. 21. The chromatography apparatus as claimed in claim 20 wherein said third and fourth positions are the same as said first and second positions. 22. A method of performing liquid chromatography with a chromatography column, substantially as herein described with reference to the accompanying drawings. 23. A chromatography apparatus, substantially as herein described with reference to the accompanying drawings.

Documents:

626-mas-1997-abstract.pdf

626-mas-1997-claims duplicate.pdf

626-mas-1997-claims original.pdf

626-mas-1997-correspondance others.pdf

626-mas-1997-correspondance po.pdf

626-mas-1997-description complete duplicate.pdf

626-mas-1997-description complete original.pdf

626-mas-1997-drawings.pdf

626-mas-1997-form 1.pdf

626-mas-1997-form 26.pdf

626-mas-1997-form 3.pdf

626-mas-1997-other documents.pdf