Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
1999-10-29
2001-10-09
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S656000, C210S101000, C073S061560
Reexamination Certificate
active
06299767
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to high pressure capillary liquid chromatography, and more particularly to a method and apparatus for high-pressure delivery of solvent or solvent mixtures, such as is useful in the art of liquid chromatography.
BACKGROUND OF THE RELATED ART
The practice of HPLC generally requires that a molecular species to be separated or analyzed be dissolved in a liquid, the mobile phase, and conveyed by that liquid through a stationary phase. In the stationary phase, a large surface area is presented which is in intimate contact with the mobile phase. Mixtures of analyte compounds, dissolved in the mobile phase, can be separated during passage through the column by processes of adsorption or retention, which act differently on the various analyte species. The differential retention causes the analytes to elute from the column with respect to time and volume. The eluting analytes will typically transit through an in-line detector, where quantitative and/or qualitative examination of analytes will occur.
High pressure liquid chromatography (HPLC) solvent delivery systems are used to source either single-component liquids or mixtures of liquids at selected pressures which can range from substantially atmospheric pressure to pressures on the order of ten thousand pounds per square inch. The above pressures are required to force the mobile phase through the fluid passageways of a stationary phase support, where separation of dissolved analytes can occur. The stationary phase support may comprise a packed bed of particles, a membrane or collection of membranes, a microfabricated structure typically comprising an array of fluid passageways etched into a solid support, or an open column or tube.
Often, analysis conditions require the mobile phase composition to change over the course of analysis which is termed “gradient elution”. In gradient elution, the viscosity of the mobile phase may change and the pressure necessary to maintain the required volumetric flow rate will change accordingly.
The elution behavior of analyte molecules is a function of the characteristics of both the stationary and mobile phases. To the extent that the properties of the stationary phase may remain substantially fixed throughout the analysis, variation in elution behavior is predominantly a result of variation in the properties of the mobile phase. In an “isocratic mode” of chromatography, the solvent composition remains substantially constant as a function of time, and analytes in the sample will tend to elute when a prescribed mobile phase volume has transited the column. In a “gradient mode” of chromatography, the solvent composition is required to change as a function of time, tracking a user defined profile. In this mode, analytes will elute in response to both the composition and the volume of solvent delivered.
The separation process occurring in liquid chromatography can result in the separation of an injected sample mixture into its component parts. These component parts are eluted from the column in reasonably distinct zones or bands. As these bands pass through a detector, their presence can be monitored and a detector output can be produced. This output includes a pattern of analyte concentration within the eluting bands, which can be represented by means of a time varying electric signal, and gives rise to the nomenclature of a “chromatography peak.” These peaks may be characterized with respect to their retention time, that is, the time in which the center of the band transits the detector relative to the time of injection. In many applications, the retention time of a peak is used to infer the identity of the eluting analyte based upon related analyses incorporating standards or calibrants. The retention time of a peak is strongly influenced by the mobile phase composition and by the volume of mobile phase, which has passed over the stationary phase.
The utility of chromatography relies heavily on run-to-run reproducibility, such that a given analysis can be compared with an analysis of standards or calibrants with confidence in the resulting data. Known pumping systems exhibit some non-ideal characteristics which result in diminished separation performance and diminished run-to-run reproducibility.
Among the non-ideal pump characteristics exhibited in known pumping systems are, generally, fluctuations in solvent composition and/or fluctuations in volumetric flow rate. Such volumetric flow rate fluctuations in present and known HPLC pumping systems disadvantageously cause varying retention times for a given analyte. That is, the amount of time that an analyte is retained in the stationary phase fluctuates undesirably as a function of the undesirable volumetric flow rate fluctuations. This creates difficulties in inferring the identity of a sample from the retention behavior of the components. Volumetric flow rate fluctuations can result in fluctuations in solvent composition when the output of multiple pumps is summed to provide a solvent composition.
Fluctuations in solvent composition in present and known HPLC systems disadvantageously result in interactions with the systems analyte detector and produce perturbations which are detected as if they arose from the presence of a sample. In effect, an interfering signal is generated. This interfering signal is summed with the actual signal attributable to the analyte, producing errors when the quantity of an unknown sample is calculated from the area of the eluting sample peak.
In light of the above, the requirements imposed on HPLC solvent delivery systems are severe. HPLC pumps are typically required to deliver solvents at pressures which can range from several pounds per square inch to as much as 10,000 pounds per square inch. There are problems and non-ideal effects associated with delivering liquids for chromatography against elevated pressures including seal deformation under load and absolute seal leakage. HPLC pumps are expected to output the mobile phase solvent at precisely controlled flow rates in a smooth and uniform manner. In the case of gradient chromatography or in the case of isocratic chromatography, where a fixed solvent composition is blended in real time during the separation, there is the further requirement that mobile phase composition as well as flow rate be precisely and accurately controlled during delivery. However, system operating pressures may be changing very substantially during the separation and the compressibilities of the constituent mobile phase solvents may be quite different.
The large errors associated with the compression or relaxation of large volumes of fluid can be minimized by the use of small volume syringe pumps that utilize multiple syringe strokes to deliver solvent through the course of a chromatographic separation period. However, these pumps typically suffer from flow perturbations associated with the transition of fluid delivery from one syringe cycle to the next.
An emerging area of chromatographic separation and analysis is developing around the use of extremely narrow bore separation columns. Such columns have been termed “capillary columns” with diameters typically in the range of 30 to 800 micron internal diameter. Such columns may be packed with a particulate packing material, or in the smallest diametral range, the stationary phase may be provided by the column wall itself or a coating applied to that wall. Mobile phase flow rates for such particulate packed capillary columns can typically range from approximately one nanoliter per minute to ten or more microliters per minute. These figures represent a three to six order-of-magnitude reduction in flow rate and consequently a similar reduction in the volume of the separation from what is currently practiced on, for example, the four millimeter internal diameter columns widely commercially available. HPLC systems designed around capillary columns have particular utility when the HPLC separation is coupled with a downstream process that does not readily tolerate large amounts of HPLC mobile p
Janiuk Anthony J.
Michaelis Brian
Therkorn Ernest G.
Waters Investments Limited
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