Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-06-07
2001-11-06
Houtteman, Scott W. (Department: 1656)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C096S103000
Reexamination Certificate
active
06312575
ABSTRACT:
FIELD OF THE INVENTION
This invention relates in general to the field of analysis of chromatograms and more particularly, though not limited to, determining chromatographic variability in order to increase the accuracy of chromatographic analysis.
BACKGROUND OF THE INVENTION
Chromatography is a technique widely used in the analysis of multi-component substances. In chromatography, a liquid or gas, of known or unknown composition, is injected into a chromatograph which generates a chromatogram in the form of a two dimensional graph in which absorptivity of the injected liquid, or conductivity of the injected gas or some other physical response is plotted against time. The absorptivity of the liquid or conductivity of the gas with respect to time as it passes through the chromatograph is indicative of the composition of the liquid or gaseous mixture.
Common uses of chromatography include quality control, in which manufactured substances are analyzed to verify the composition, and qualitative and quantitative analysis in which chromatograms derived from unknown substances are generated to analyze and determine the composition of the substance. In a quality control application, a chromatogram of a known and desired substance is generated and compared to the chromatogram of the manufactured substance. In qualitative and quantitative analysis applications, one or more chromatograms are generated of the unknown substance in an attempt to identify the components or quantify the amounts of each of the components of the substance.
In either of the above applications, the chromatogram(s) must be analyzed to determine similarities or differences with other chromatograms. Typically, such analysis requires analysis and comparison of the peaks, including the retention time, height and area of peaks between one chromatogram with those of another. In order to perform such a comparison, a method of identifying the peaks to be compared must be developed and optimized, and then a method to compare the peaks and other above identified aspects of the chromatograms must be developed. Principal Component Analysis (PCA) is one such technique and is referred to by G. Malmquist and R. Danielsson in a paper entitled “Alignment of Chromatographic Profiles for Principal Component Analysis: A Prerequisite for Fingerprinting Methods”,
Journal of Chromatography A,
687 (1994) 71-88. As described by Malmquist and Danielsson, retention times of selected peaks are used to align corresponding chromatograms. Once they are aligned, the absorbances are themselves compared. Another technique is described by J. P. Mason et al. in an article entitled “A Novel Algorithm for Chromatogram Matching in Qualitative Analysis”,
Journal of high Resolution Chromatography
, v. 15, pp. 539-547 (August 1992) which describes an automated chromatographic matching technique which compares only peak heights, areas and retention times.
Chromatographic analysis must also take into account variability introduced by the chromatograph such as baseline drift, retention time wander and concentration change. Such variations of the chromatograph are manifested as variations in the chromatograms generated by the chromatograph and further complicate the analysis by requiring the analysis to take into account those several variations. In the above referenced paper by Malmquist and Danielsson, a technique is described for compensating for chromatographic variability in the context of enhancing chromatographic analysis by PCA
Known techniques for chromatographic analysis such as described by Malmquist and Danielsson and Mason et al. typically require the steps described above of specifying a method of identifying peaks to be compared, specifying a method of comparing the various aspects of the peaks, and then actually performing the comparisons, while taking into account the effects of chromatographic variability. Although computerized techniques such as those described by Mason are helpful in performing such tasks, many known techniques continue to be time consuming, sometimes tedious and require the skills of highly trained personnel.
It is accordingly an object of the present invention to provide a computation devices or systems for chromatographic analysis which compensate for chromatographic variability and which perform chromatographic analysis without requiring the characterization of peaks or other chromatographic features as required by known techniques.
SUMMARY OF THE INVENTION
Embodiments of the present invention are directed to devices and methods for performing chromatographic analysis. As used herein, the term “chromatographic analysis device” refers to computers and computational apparatus for receiving and processing data; and, software and programming which directs such computers and computational apparatus for receiving and processing data.
In accordance with the primary object of the invention, a chromatographic analysis method employs a chromatogram alignment procedure which characterizes variability of a chromatograph. In accordance with a further object of the invention, the chromatographic analysis method employs the characterized chromatographic variability in the comparison of chromatograms. Advantageously, the chromatographic analysis method of the present invention compares chromatograms as patterns and does not require the characterization of peaks or the identification of the accompanying peak lift-off or touch down points. Moreover, peak heights, areas or retention times need not be computed. The phrase “characterization of peaks” is intended to refer to the process of peak integration which requires identification of the baseline by identification of the lift and touchdown points of the peak in question and then actual integration of the peak. The chromatographic pattern analysis method of the present invention applies to chromatograms obtained by isocratic or gradient chromatographic separations. In isocratic chromatography, the composition of the solvent is held constant throughout the chromatographic separation. In gradient chromatography, the composition of the solvent is varied in a predetermined way to obtain enhanced control over the retention times of compounds.
In a first aspect, a chromatographic analysis method operating in accordance with the principles of the present invention determines differences between a standard chromatogram and a sample chromatogram. The standard chromatogram is represented by a set of standard data points, indicative of the standard chromatogram over a selected elution time range and the sample chromatogram is represented by a set of sample data points indicative of the sample chromatogram over the same elution time range or an elution time range offset by a fixed amount. The standard data points and the sample data points are each generated by sampling each respective chromatogram at a fixed rate. The chromatographic analysis method then generates a plurality of sets of chromatographic variability data points from the standard data points, each of the sets of chromatographic variability data points being indicative of effects of a predetermined source of chromatographic variability on the standard chromatogram. The method also generates a set of modified standard data points, which correspond to the standard data points modified as a function of the chromatographic variability data points, to model chromatographic variability of a chromatograph which generates said chromatograms.
The method described above may be used with standard and sample chromatograms generated from the same mixtures in order to determine variability of the chromatograph. In addition, the method may be used to determine similarities or differences between the standard chromatogram and sample chromatograms from different or unknown mixtures. To assist in such a comparison, the method, in a second aspect, generates residue values which are indicative of differences between the standard chromatogram and the sample chromatograms. The residue value generated from a comparison of the standard to sample chromatog
Houtteman Scott W.
Michaelis Brian L.
Waters Investments Limited
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