Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
1999-10-01
2001-10-02
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S659000, C210S143000, C210S198200, C073S061520, C436S161000
Reexamination Certificate
active
06296771
ABSTRACT:
BACKGROUND OF INVENTION
The present invention generally relates to liquid chromatography, and specifically, to high-pressure liquid chromatography (HPLC) methods and systems for rapidly separating and/or characterizing a plurality of samples. The invention particularly relates, in a preferred embodiment, to hybrid parallel-serial HPLC methods and systems for separating and/or characterizing a combinatorial library comprising different polymers.
Liquid chromatography is generally well known in the art. High-pressure liquid chromatographic techniques involve injection of a sample into a mobile-phase that flows through a chromatographic column, separation of one or more components of the sample from other components thereof in the chromatographic column, and detection of the separated components with a flow-through detector. Approaches for liquid chromatography typically vary, however, with respect to the basis of separation and with respect to the basis of detection.
Gel permeation chromatography (GPC), a well-known form of size exclusion chromatography (SEC), is a frequently-employed chromatographic technique for separation of samples generally, and for polymer size determination particular. Another chromatographic separation approach is illustrated by U.S. Pat. No. 5,334,310 to Fréchet et al. and involves the use of a porous monolithic stationary-phase as a separation medium within the chromatographic column, combined with a mobile-phase composition gradient. Other separation approaches are also known in the art, including for example, normal-phase (e.g., adsorption) chromatography and reverse-phase chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, ion-exchange chromatography, affinity chromatography, among others.
After separation, a detector can measure a property of the sample or of a sample component—from which one or more characterizing properties, such as molecular weight can be determined as a function of time. Specifically for polymers, for example, a number of molecular-weight related parameters can be determined, including for example: the weight-average molecular weight (M
w
), the number-average molecular weight (M
n
), the molecular-weight distribution shape, and an index of the breadth of the molecular-weight distribution (M
w
/M
n
), known as the polydispersity index (PDI). Other characterizing properties, such as concentration, size (e.g. for particles or polymers), architecture, chemical composition and/or chemical composition distribution can likewise be determined. A variety of continuous-flow detectors have been used for measurements in liquid chromatography systems. Common flow-through detectors include optical detectors such as a differential refractive index detector (RI), an ultraviolet-visible absorbance detector (UV-VIS), or an evaporative mass detector (EMD)—sometimes referred to as an evaporative light scattering detector (ELSD). Additional detection instruments, such as a static-light-scattering detector (SLS), a dynamic-light-scattering detector (DLS), and/or a capillary-viscometric detector (C/V) are likewise known for measurement of properties of interest.
Broadly available liquid chromatography systems are not entirely satisfactory for efficiently screening larger numbers of samples. With respect to polymers, for example, high-performance liquid chromatographic techniques can typically take up to an hour for each sample to ensure a high degree of separation over the wide range of possible molecular weights (i.e., hydrodynamic volumes) for a sample. Notably, however, substantial improvements in sample throughput have been achieved in the art. For example, rapid-serial approaches for characterizing polymers have been developed by Symyx Technologies, Inc. (Santa Clara, Calif.) and disclosed in the aforementioned co-pending U.S. patent applications from which the present application claims priority. As another example, U.S. Pat. No. 5,783,450 to Yoshida et al. discloses rapid-serial protocols and systems for preparation, purification and separation of small molecules such as catecholamines and protaglandins from biological samples such as blood.
Parallel approaches for liquid chromatography have also been contemplated in the art. Zeng et al.,
Development of a Fully Automated Parallel HPLC/Mass Spectrometry System for the Analytical Characterization and Preparative Purification of Combinatorial Libraries, Anal. Chem
. 70, 4380-4388 (1998), disclose analytical and preparative HPLC methods and systems involving the sequential preloading of samples onto two chromatographic columns, and then applying a mobile-phase in parallel to each of the columns to effect parallel separation of the samples. According to an alternative approach disclosed in U.S. Pat. No. 5,766,481 to Zambias et al., parallel separation of a plurality of molecules is effected by forming a mixture of selected, compatible molecules, and subsequently resolving the mixture sample into its component molecules by separation in a single-channel HPLC system. Parallel approaches have likewise been employed in other separation protocols, such as capillary electrophoresis. See, for example, U.S. Pat. No. 5,900,934 to Gilby et al.
Although such parallel approaches and systems have been generally contemplated, there nonetheless exists a need in the art for improving such approaches and systems with respect to overall sample throughput and/or quality of data. Moreover, with the development of combinatorial materials science techniques that allow for the parallel synthesis of libraries comprising a vast number of diverse industrially relevant materials, and especially polymeric materials, there is a need for HPLC methods and systems to rapidly characterize the properties of samples from such combinatorial libraries.
SUMMARY OF INVENTION
It is therefore an object of the present invention to provide HPLC systems and protocols having a higher overall sample throughput, and in preferred applications, employing such systems and protocols for characterizing combinatorial libraries of material samples such as polymer samples, and particularly, libraries of or derived from reaction mixtures such as polymerization product mixtures, to facilitate the discovery of commercially important materials such as polymeric materials, catalysts, polymerization conditions and/or post-synthesis processing conditions.
Briefly, therefore, the present invention is directed to methods for separating and characterizing components of a plurality of samples with a high-performance liquid chromatography system. According to one preferred method, a mobile phase is supplied (e.g., pumped) in parallel through each of first and second chromatographic columns of a liquid chromatography system. First and second samples are serially injected into the mobile phase of the first and second chromatographic columns, respectively. At least one sample component of the injected first and second samples is then separated from other sample components thereof in the respective chromatographic columns. Preferably, in applications to analytical chromatography, a property of at least one of the separated sample components of the first and second samples is detected. A property of interest can then be determined from the detected property (e.g., by correlation to known standards for the property of interest).
The invention is also directed to several preferred variations of the aforedesribed method. In one preferred variation, four or more different samples are serially and distributively injected into a mobile phase being supplied in parallel to four or more chromatography columns. In another preferred variation of such method, ten or more different samples are serially loaded into an injection system (and preferably into an injector such as an injection valve), and then serially and distributively injected through a multi-port switching valve into one of the mobile phases being supplied in parallel to four or more chromatography columns. In each of the aforementioned methods, the number of parallel chr
Symyx Technologies Inc.
Therkorn Ernest G.
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