Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2000-09-26
2002-08-20
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S659000, C210S143000, C210S198200, C073S061520, C436S161000, C422S070000
Reexamination Certificate
active
06436292
ABSTRACT:
BACKGROUND OF INVENTION
The present invention generally relates to liquid chromatography, and specifically, to high-pressure liquid chromatography (HPLC) methods for rapidly characterizing a plurality of samples. The invention particularly relates, in a preferred embodiment, to parallel HPLC methods for 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 particularly. Another chromatographic separation approach is illustrated by U.S. Pat. No. 5,334,310 to Frechet 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 adsorption chromatography, and reverse-phase chromatography.
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, with respect to polymer samples, for example, a number of molecular-weight related parameters can be determined, including for example: the weight-average molecular weight (Mw), the number-average molecular weight (Mn), the molecular-weight distribution shape, and an index of the breadth of the molecular-weight distribution (Mw/Mn), known as the polydispersity index (PDI). Other characterizing properties, such as mass, particle size, composition or conversion 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.
Detection methods involving post-separation treatment are known in the art. With respect to polymer samples, for example, European Patent EP 675 356 B1 to Staal discloses a method and a system for precipitating polymer components of a sample in the effluent stream from a chromatographic column, with optical detection of the precipitated components, and is hereby incorporated by reference for all purposes. Significantly, the application of such detection methods to parallel HPLC systems was not contemplated, and the benefits thereof were not heretofore appreciated in the art.
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 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 and systems for characterizing a plurality of samples by liquid chromatography or, in some embodiments, by flow-injection analysis. According to the methods, a mobile phase is supplied in parallel through each of first and second chromatographic columns of a high-pressure liquid chromatography system. First and second samples are injected into the mobile phase of the first and second chromatographic columns, respectively. In chromatographic applications, at least one sample component of the injected first and second samples is separated from other sample components thereof in the respective chromatographic columns. Significantly, after separation, but before detection, at least one separated sample component of the first and second samples is treated to change a property of at least one separated sample component thereof. The treatment is preferably precipitation and/or derivitization. A property of the treated sample component of the first and second samples is detected.
The present invention provides substantial advantages over known approaches for parallel liquid chromatography systems. High overall throughput is achieved with a parallel HPLC system—even for samples (e.g., many types of polymers) having components that would otherwise be difficult to detect, and moreover, in a cost-effective manner. These advantages are particularly realized
Symyx Technologies Inc.
Therkorn Ernest G.
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