Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2003-04-11
2004-08-03
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C073S061590, C210S198200
Reexamination Certificate
active
06770876
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method and apparatus useful in high pressure liquid chromatography (HPLC), and more particularly to an autosampler device useful for the automated introduction of small sample volumes into a HPLC system.
BACKGROUND OF RELATED TECHNOLOGY
It is desirable to identify low abundant proteins from biological samples in order to investigate the role of individual proteins in biological processes, such as in the field of proteomics. It is further desirable to develop sensitive analytical methods which provide not only the means to analyze extremely low abundant proteins but also the possibility to reduce or avoid costly and time-consuming protein enrichment processes.
One such method involves the use of HPLC. Generally, HPLC requires that a molecular species to be separated and analyzed is dissolved in liquid (the mobile phases), and then conveyed by those liquids through a stationary phase. In the stationary phase, a large surface area is presented which is in intimate contact with the mobile phases. Mixtures of analyte compounds, dissolved in the mobile phases, can be separated. The differential retention causes the analytes to elute from the column with respect to time. The eluting analytes will typically transit through an in-line detector, where quantitative and/or qualitative examination of analytes will occur. Such examination is typically performed using a mass spectrometer (MS). In recent years, the use of MS with capillary electrophoresis (CE) or liquid chromatography (LC) has become increasingly popular for analyzing low abundance proteins.
A key element in the successful analysis of low abundant proteins is to ensure that an entire protein digest at about 20 &mgr;l is injected into a capillary column with minimum sample loss. CE has been demonstrated to inject the volume of protein digest by constructing a segment of solid phase extraction material (Figeys, et al.,
J. Chromatogr
. A., 763:295-306 (1997); Figeys, et al.,
Electrophoresis,
19:2338-47 (1998)) or a piece of membrane (Naylor, et al. al.,
J. Biomed. Chromatogr.,
10:325-30 (1996)) for sample enrichment and subsequently eluting the protein digest for separation and MS analysis. On the other hand, capillary LC loads the protein digest directly into a capillary column by a pressurized sample introduction device (Shelly, et al.,
Analytical Chemistry,
56:2990-2 (1984)). While both CE/MS and capillary LC/MS achieve excellent detection limits for protein analysis, the capillary LC/MS approach is more robust, and therefore more widely used for analysis of low abundant proteins than the CE/MS method.
Known devices used for low abundant protein analysis by capillary LC/MS generally consist of a cylinder and a top cover and are capable of accommodating only one sample. Although such devices are capable of delivering a sample into a capillary LC column, the whole sample introduction procedures are manually driven and tedious. For example, in order to introduce a sample, an operator must open a stainless steel cylinder to place a sample vial in, fasten bolts through a cover, and pressurize the cylinder for sample introduction. As these operations are off line, the operator also needs to depressurize the device after loading, remove the column from the cylinder, and put the column on a LC/MS system for analysis. As a result, it is impossible to perform automated data acquisition for analysis of a batch of samples using such devices.
Known devices for automated capillary LC sampling have numerous disadvantages for analysis of low abundant proteins. For example, in order to introduce a sample volume of 20 &mgr;l into the capillary column, it is often necessary to have up to 50 &mgr;l of sample in the sample vial. As a result of this sample waste, such devices are not optimized for identifying low abundant proteins efficiently.
Accordingly, a need exists for a device useful for automated capillary LC sampling which overcomes these problems. The present invention is directed to meeting these and other needs.
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Figeys, D. et al., “Identification of proteins by capillary electrophoresis—tandem mass spectrometry: Evaluation of an on-line solid-phase extraction device”, Journal of Chromatography A, vol. 763, pp. 295-306 (1997).
Figeys, D. et al., “Optimization of solid phase microextraction—capillary zone electrophoresis—mass spectrometry for high sensitivity protein identification”, Electrophoresis, vol. 19, pp. 2338-2347 (1998).
Naylor, S. et al., “Membrane Preconcentration—Capillary Electrophoresis—Mass Spectrometry in the Analysis of Biologically Derived Metabolites and Biopolymers”, Biomedical Chromatography, vol. 10, pp. 325-330 (1996).
Shelly, D.C. et al., “Dead-Volume Free Termination for Packed Columns in Microcapillary Liquid Chromatography”, Anal. Chem., vol. 56, pp. 2990-2992 (1984).
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Allen John W.
Gu Ming
Li Wenjeng
Bristol--Myers Squibb Company
Hughes James P.
Lange Keith R.
Lee John R.
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