Measuring and testing – Liquid analysis or analysis of the suspension of solids in a... – Content or effect of a constituent of a liquid mixture
Reexamination Certificate
2000-04-20
2001-11-20
Larkin, Daniel S. (Department: 2856)
Measuring and testing
Liquid analysis or analysis of the suspension of solids in a...
Content or effect of a constituent of a liquid mixture
C073S061560, C210S659000, C210S198200, C422S070000
Reexamination Certificate
active
06318157
ABSTRACT:
BACKGROUND OF THE INVENTION
High performance liquid chromatography (HPLC) is a versatile technique that allows for separation of compounds with molecular weights ranging from approximately 54 to greater than 450,000 for analysis and/or identification. Detection limits are dependent on the type of detector and may range from femtograms to micrograms at the analytical scale, to milligrams at the semipreparative scale, and to grams at the preparative scale. The technique is relatively robust having no requirements for volatile compounds or derivatives and allows for the analysis of thermally labile compounds. Samples with wide ranging polarities can be analyzed in a single analysis. HPLC provides relatively fast analysis times, reproducibility, and sensitivity. Typical run times (analysis time) range from 5 to 30 minutes but can be longer than an hour for gradient runs. Run times under a minute have been achieved on microbore columns, but more often than not, these fast separations have limited applications because less than optimal chromatographic separations are achieved.
High performance liquid chromatography coupled with mass spectrometry (MS) is common for quantitative analysis, confirmation of identity, and structural determination of unknowns. Relatively recent developments in mass spectrometry, especially, electrospray ionization (ESI) and atmospheric pressure ionization (API), have made the coupling a useful and reliable means for routine liquid chromatography/mass spectrometers (LC/MS) sample analysis. Specifically, tandem MS (LC/MS/MS) provides very high selectivity and specificity and as a result has become a powerful tool for pharmaceutical analyses and quantization. Although LC coupled with MS/MS has become an indispensable tool for pharmacological analysis by providing high sensitivity along with relatively fast analysis times, there still is an increased demand for higher sample throughput than is provided today. Currently, an analytical bottleneck exists.
In addition, drug discovery processes have been greatly accelerated by advances in combinatorial chemistry. The analytical demands created by modern drug discovery practices require improved sample preparation and analysis capabilities. Combinatorial chemical syntheses are performed in parallel whereas LC analyses used to characterize the products of parallel syntheses are currently performed in a serial fashion thus creating an analytical bottleneck. Because of the need for high sample throughput, chromatographic separations are often sacrificed in order to decrease analysis times. Although faster analyses are achieved, often it leads to compromised chromatographic separation resulting in lower accuracy and precision.
The major disadvantage of serial LC analysis is the inherent “idle time” that occurs during pre- and post-analyte elution. Here, idle time refers to the time during the analytical run that the analyte is not being detected; for example, injection time and preparation, elution time of non-analyte eluting peaks, column wash cycles, and column equilibration. Typically in an HPLC run, the analytes of interest elute in a short time relative to the total run time of the analytical run. To date, the most common approach taken is to reduce chromatography separation times as a means of increasing sample throughput. This approach will inherently be limited because it is conducted in a serial fashion whereas the syntheses are conducted in a parallel fashion.
U.S. Pat. No. 4,364,263 to Sankoorkal et al discloses a high pressure liquid chromatographic system including a plurality of parallel columns with a valving system which directs one solvent/sample flow to a single column and then to a detector.
U.S. Pat. No. 3,373,872 to Hodina discloses a chromatography apparatus where a two way valve selectively connects one of two columns to a solution pump.
Several different approaches have been taken to achieve parallel chromatography and in order to increase throughput. de Biasi et al. proposed a four-channel multiplexed electrospray interface used in combination with four LC columns and multi-syringe probe injector, such as a Gilson Multiprobe. This interface rapidly switches between multiple liquid streams which results in lower dwell times and reduced sensitivity. [de Biasi et al.,
Rapid Communication In Mass Spectrometry,
1999, Vol. 13, pgs. 1165-1168] The approach may be useful for unknowns where sensitivity is not an issue, but would it be useful for PK analysis where sensitivity is of great importance. Also, this approach uses a specific multiprobe injector system that contains multiple injectors with many valves and mechanical parts. Additionally, because this approach requires modifications to the mass spectrometer ion source, the user is limited to a specific instrument vendor.
Another approach was taken by Korfmacher et al. who used two separate LC pumping systems, two autosamplers, and two columns connected via a divert valve to a single mass spectrometer. A major disadvantages of this system is that it requires a significant investment in capital equipment as it contains the HPLC hardware of two conventional systems. [Korfacher, 1999]
Zeng et al. [
Analytical Chemistry,
1998, Vol. 70, pgs. 4380-4388] have shown parallel separations using two columns and an autosampler equipped with multiple injectors (multiprobe) for automated characterization of and purification of combinatorial libraries. This system operates two analytical or preparative columns in parallel through a valving system and dual electrospray ionization interface. The design of this dual interface permits the ionization of the analytes in one line to impact the ionization of the analytes in the other, and an additional disadvantage of this system is that two isobaric analytes cannot be quantified simultaneously. This injector system employed contains eight separate injectors each with a corresponding sample probe (the authors used two for this work). The system was configured so that the autosampler's two injectors simultaneously admitted the samples into two different corresponding columns and then to a dual sprayer ionspray interface followed by introduction into a single mass spectrometer. The authors system works for qualitative applications where molecular weights are known prior to analyses while also having a requirement of varying m/z values in the analyses. However, for instances where same transition m/z value is desired for many samples, such as the case for quantitative analyses, MS can only measure one ion beam at a time on current instrumentation. There are several disadvantages to the system described above: 1. The high numbers of mechanical parts (i.e. valves and injector probes) increase the rate of mechanical failure. 2. The additional tubing lengths needed to connect the numerous valves contribute to increased dead volume and swept volume and resulting in decreased chromatographic resolution. 3. Because separate probes (injector needles and syringe barrels) inject samples into separate valves, each with separately plumbed lines, greater variability results to that of a single injector system (fewer parts). 4. The inherent cost of the system is increased because of the increased hardware and complexity of the system. In addition, a user is limited to this particular manufacturer and specific system.
SUMMARY OF THE INVENTION
It is a primary object of the present invention to provide a novel and improved high-throughput parallel liquid chromatography system and method utilizing “staggered” parallel chromatography in order to admit analytes sequentially from a multiplicity of columns into a detector.
Another object of the present invention is to provide a novel and improved high-throughput parallel liquid chromatography system using a single injector and a plurality of parallel columns to admit analytes to a single detector. A significant increase in analysis speed is achieved because analytes eluding from a given column are directed to the detector only during elution times.
Yet anoth
Corso Thomas N.
Van Pelt Colleen K.
Advanced Bioanalytical Services, Inc.
Goldman Michael L.
Larkin Daniel S.
Nixon & Peabody LLP
Sixbey Daniel W.
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