Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2002-08-28
2004-05-04
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S659000, C210S143000, C210S198200
Reexamination Certificate
active
06730228
ABSTRACT:
BACKGROUND OF INVENTION
The present invention generally relates to methods and apparatus for characterization of polymer samples in liquid chromatography systems, and specifically, for characterization of polymer samples in multi-dimensional liquid chromatography systems. The invention particularly relates, in a preferred embodiment, to characterization of polymer samples in a comprehensive, directly-coupled, multi-dimensional high-performance liquid chromatography systems including a first HPLC dimension adapted for determining composition (e.g., adapted for reverse phase chromatography, adsorption chromatography, and the like such as mobile phase gradient-elution chromatography) and a second HPLC dimension adapted for determining molecular weight or size (e.g., adapted for size exclusion chromatography such as gel permeation chromatography).
Multi-dimensional high-performance liquid chromatography systems are known in the art. See e.g., Murphy et al.,
Effect of Sampling Rate on Resolution in Comprehensive Two
-
Dimensional Liquid Chromatography
, Anal. Chem. 70, 1585-1594 (1998); Murphy et al.,
One
-
and Two
-
Dimensional Chromatographic Analysis of Alcohol Ethoxylates
, Anal. Chem. 70, 4353-4360 (1998); Kilz et al.,
Two Dimensional Chromatography for the Deformulation of Complex Copolymers
, Chapter 17, pp. 223-241 of the text entitled “Chromatographic Characterization of Polymers, Hyphenated and Multidimensional Techniques”, edited by Provder et al. (American Chemical Society, Advances in Chemistry Series 247, 1995); Opiteck et al.,
Two
-
Dimensional SEC/RPLC Coupled to Mass Spectrometry for the Analysis of Peptides
, Anal. Chem. 69, 2283-2291 (1997); and Trathnigg et al.,
Two
-
Dimensional Liquid Chromatography of Functional Polyethers
, Chapter 13, pp. 190-199 of the text entitled “Chromatography of Polymers, Hyphenated and Multidimensional Techniques”, edited by Provder et al. (American Chemical Society, Symposium Series 731, 1999), each of which is hereby incorporated by reference for all purposes.
Although the methods and systems disclosed to date in the art have proven to be useful for characterizing biological and non-biological polymer samples, they generally suffer from inefficiencies with respect to overall sample throughput, and/or with respect to complicated control and/or operation schemes and systems.
Accordingly, there remains a need in the art for improved methods and systems for effecting multi-dimensional liquid chromatography for characterization of polymer samples.
SUMMARY OF INVENTION
It is therefore an object of the present invention to provide methods and apparatus that allow for more efficient, and relatively less complicated approaches than the prior art for characterizing polymer samples, and especially for fingerprinting polymer samples such as non-biological copolymer samples.
Briefly, therefore, the present invention is directed, generally, to methods for characterizing a polymer sample in a multi-dimensional liquid chromatography system. In preferred embodiments, the invention is directed to methods for characterizing a library of polymer samples in a multi-dimensional liquid chromatography system. The multi-dimensional liquid chromatography system comprises at least a first dimension and a second dimension, and in some embodiments, can include a third dimension, a fourth dimension and/or additional dimensions. Preferably, each of the first dimension and the second dimension is a high-performance liquid chromatography (HPLC) subsystem. The multi-dimension liquid chromatography system is preferably a comprehensive multi-dimension liquid chromatography system wherein at least a portion of each of the sample components separated in the first dimension are further separated into subcomponents in the second dimension. Further, the first dimension and second dimension of the multi-dimensional liquid chromatography system are preferably directly-coupled, wherein the components separated in the first dimension are sampled in near real time (e.g., in-line) as they elute off of the first-dimension chromatography column(s) for injection into the second dimension—for example, through a second-dimension in-line multi-port injection valve.
The method generally comprises, for characterization of a single polymer sample, injecting the polymer sample into a first-dimension high-performance liquid chromatography subsystem, separating the polymer sample into two or more components in the first-dimension liquid chromatography subsystem, optionally detecting a property of the first-dimension separated components in the first-dimension eluent (e.g., using a flow-through detector), sampling at least a portion of each of the first-dimension separated components for directly-coupled injection into a second dimension, injecting each of the sampled portions into a second-dimension high-performance liquid chromatography subsystem, separating at least one of, and preferably each of the sampled portions of the first-dimension separated components into two or more subcomponents in the second-dimension liquid chromatography subsystem, and detecting a property of the second-dimension separated subcomponents in the second-dimension eluent (e.g., using a flow-through detector).
More specifically, for characterizing a single polymer sample, the polymer sample is injected (e.g., using a multi-port injection valve as a first-dimension injector) into a first-dimension mobile phase of a first HPLC dimension of the multi-dimensional liquid chromatography system. At least one sample component of the injected polymer sample is chromatographically separated from other sample components thereof in a first-dimension liquid chromatography column (e.g., in selectable fluid communication with the first-dimension injector), such that a first-dimension mobile phase eluent from the first-dimension column comprises two or more first-dimension separated sample components. Optionally, a property of the first-dimension separated components in the first-dimension mobile phase effluent can be detected using a flow-through detector (e.g. mass detector, universal concentration detector, light-scattering detector, etc.). Then, at least a portion of each of the first-dimension separated sample components from the first-dimension mobile phase eluent are sampled for directly-coupled injection into a second HPLC dimension of the multi-dimensional chromatography system (e.g., using sample loops associated with a multi-port injection valve). The sampled portions of each of the first-dimension separated sample components are then injected directly into a second-dimension mobile phase of the second HPLC dimension of the multi-dimensional liquid chromatography system (e.g., using a multi-port injection valve as a second-dimension injector). At least one subcomponent of the injected sample portions is chromatographically separated from other subcomponents thereof in a second-dimension liquid chromatography column (e.g., in selectable fluid communication with the second-dimension injector), such that a second-dimension mobile phase eluent from the second-dimension column comprises two or more second-dimension separated subcomponents for one or more, and in some cases, for each of the sampled portions of each of the first-dimension separated sample components. A property of the second-dimension separated subcomponents are detected in the second-dimension mobile phase effluent using a flow-through detector.
For characterization of a library of polymer samples comprising four or more polymer samples, the aforementioned steps, as generally or specifically characterized, of injecting into the first dimension, separating into components in the first dimension, optionally detecting separated components in the first-dimension eluent, injecting into the second dimension, separating into subcomponents in the second dimension and detecting separated subcomponents in the second-dimension eluent are repeated for each of the polymer samples of the library.
In preferred embodiments, the method is further characterized acco
Carlson Eric D.
Nguyen Son Hoai
Petro Miroslav
Summa & Allan P.A.
Symyx Technologies Inc.
Therkorn Ernest G.
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