Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
1999-09-30
2001-02-20
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S656000
Reexamination Certificate
active
06190550
ABSTRACT:
This invention relates generally to chromatography and more particularly to apparatus for and methods of monitoring improved high performance liquid chromatographic systems.
BACKGROUND OF THE INVENTION
High performance liquid chromatography is effected generally by percolating a number of component solute molecules in a flowing stream of a liquid through a packed bed of particles, known as the stationary phase, which efficiently separates the various types of molecules from one another.
Such separations, particularly in the preparative area, have significant limitations typically occasioned by the batch nature of the current processes. Typically, a chromatographic column is first equilibrated by flowing a liquid 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 liquids or an eluant gradient are run sequentially through the column to release bound solute selectively. The eluted solutes, usually in relatively minute quantities, are thus temporally separated at the output of the column and the process is repeated cyclically. It is highly desirable to verify the intended changes in solution composition that actually arrive at the inlet of the chromatographic column in the intended manner. It may also be desirable to monitor the time-varying output of the column which provides an indication of band-spreading due to mixing of adjacent volumes of different solvents in the input conduits and the column itself, and malfunctions in the eluant gradient generating devices or other malfunctions which may cause undesirable behavior of the system. In chromatographic systems typical of the prior art, the electrical input used to control the gradient generator also generate the display of the gradient profile, and does not provide any measurement of the actual fluid output of the gradient generator. Consequently, malfunctions (even as common and trivial as running out of solvent) that create gradient conditions other than those intended, will only be noted when the chromatographic separation fails to yield the desired result, i.e. when it is too late to resolve.
Conventional or standard detectors are designed generally to be installed downstream from the chromatographic column where the pressure level is the lowest in the system and where the desired solute has been diluted as a result of the separation process. The material or solute, as it is detected at the outlet of the column, is generally in a much larger volume of solvent than when it is introduced at the input to the column. The greater volume of the peak to be detected at the column outlet means that band-spreading in the detector can be greater without any major effect on the overall separation, and because the more dilute conditions require a more sensitive detector, usually a larger detector cell is provided which tends to produce even more band-spreading. Since the pressure requirements imposed on detectors placed at the column output are very modest, the detector designs are slanted toward high sensitivity and it is doubtful that currently used detectors are capable of sustaining the pressure levels commonly present at the inlet to HPLC columns. Even if such detectors were strengthened to meet such pressure demands, because they tend to introduce excessive band-spreading, they would not adequately serve as detectors for the column inlet.
The foregoing considerations are, a fortiori, important in the context of certain novel methods and apparatus for performing HPLC at the very high inlet pressures required to insure that flow through the chromatographic column occurs at a reduced velocities of greater than about 5,000, a highly efficient chromatography system described more fully in U.S. Pat. Nos. 5,772,874, 5,795,469 and 5,919,368, the same being incorporated in their entirety herein by reference.
OBJECTS OF THE INVENTION
A principal object of the present invention is to provide improved monitoring for chromatographic apparatus and processes effecting high capacity, high resolution separation of solutes. Other objects of the present invention are to provide such chromatographic apparatus and processes that respond very quickly to changes in composition, that do not introduce significant band-spreading, and are particularly useful in the separation of high molecular weight products such as biologicals and the like. Yet other objects of the present invention are to provide such apparatus and processes as will enable monitoring of fluid flow through preparative chromatographic apparatus employing large pressure levels at the column input; and particularly to provide such monitoring of fluid flow in chromatographic apparatus in which reduced velocities through the column are effected at levels preferably greater than about 5000.
SUMMARY OF THE INVENTION
To these ends the present invention is directed to novel methods and apparatus for monitoring liquid chromatography apparatus, and for monitoring liquid chromatography apparatus employing relatively high inlet pressures typically characteristic of equipment employing the principles set forth in the aforesaid U.S. Pat. Nos. 5,772,874, 5,795,469 and 5,919,368. Accordingly, the invention comprises a method of monitoring performance of liquid chromatography apparatus comprising an elongated chromatography column having conduit means coupled to the column for effecting a flow of liquid through the column, in which method, changes in selected electromagnetic properties of liquid flowing through the conduit are measured. To this end, in one embodiment, the invention comprises chromatography apparatus including the usual chromatographic column together with means for controlling the injection of sample liquid and eluant liquid into and/or out of the column through the conduit means or conduit. In a preferred embodiment, the internal dimensions and configuration of the conduit and the inlet to the column are selected so that the liquid volumes of eluant and sample experience substantially no band-spreading when flowing through the device. For example, the cross-section dimensions and geometry of the conduit are selected to provide fluid flow paths at both the inlet and outlet of the column that have substantially identical dimensions and geometry. Means are coupled to the conduit for measuring selected electromagnetic properties of liquid flowing therethrough, for example, the electrical conductivity of the liquids in the conduit. Other electromagnetic properties of the liquid in the conduit that may be measured alone or in combination are the electrical capacitance, magnetic susceptibility, electrical resistance and the like. It should be understood that the liquid in the conduit, with respect to its electromagnetic properties, refers to not only the liquid per se but that liquid in combination with any solute or suspended material therein. In a preferred embodiment, the means for measuring the selected electromagnetic properties preferably comprises detector means for detecting the selected property and means for measuring the property so detected. To this end, in one embodiment, the detector means includes at least a pair of surfaces responsive to the electromagnetic properties and disposed at spaced-apart locations with respect to the conduit, the responsive surfaces being formed of electrically conductive material, with the adjacent conduit surfaces being formed of substantially electrically non-conductive or insulating material. In yet another embodiment, the means for controlling the injection of sample and eluant fluids into the column preferably serves to introduce respective volumes of the liquids into an input conduit substantially simultaneously at spatially separated locations so that the liquids travel through the column at a common group velocity as closely bunched liquid plugs.
The invention is intended in one particular embodiment to be applicable to chromatography apparatus of the type described in the aforesaid U.S. Pat. Nos. 5,772,874, 5,795,469 and 5,919,368. In su
Cohesive Technologies Inc.
Schiller & Associates
Therkorn Ernest G.
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