JP5146344B2 - Chromatographic data processor - Google Patents

Description

  The present invention relates to a data processing apparatus for processing data obtained by a chromatograph such as a liquid chromatograph (LC), and more particularly, a chromatograph using a multichannel detector such as a photodiode array detector as a detector. The present invention relates to a data processing apparatus.

  In a chromatograph such as a liquid chromatograph (LC), a chromatogram in which a peak corresponding to a component contained in a sample appears can be obtained. However, the peak appearing in a single shape appearing in the chromatogram is not necessarily due to a single component, and there may be a case where a plurality of components that are not sufficiently separated by the column overlap. Therefore, conventionally, a peak purity determination process has been performed in order to check whether or not the peak of the chromatogram is due to a single component.

  Non-Patent Document 1 discloses a technique for determining a peak purity on a chromatogram obtained by a liquid chromatograph using a photodiode array (PDA) detector. Since a PDA detector can measure an absorption spectrum over a predetermined wavelength range by a sample in a short time, a liquid chromatograph using this as a detector has three dimensions of time, wavelength, and signal intensity. Three-dimensional data can be acquired. Therefore, in the above document, peak purity determination by the following two methods is proposed.

  [Method 1] When determining the purity of a certain peak on the chromatogram, the absorption spectrum patterns at the three points of the peak top, peak rising slope, and peak falling slope are compared, and the spectral pattern and peak at the peak top are compared. The degree of coincidence with the spectrum pattern in the ascending slope and the degree of coincidence between the spectrum pattern in the peak top and the spectrum pattern in the peak descending slope are respectively obtained. Then, an average value of the two matching degrees is obtained, and whether or not it is a single peak is determined based on whether or not it is larger than a predetermined threshold value. A method for obtaining the degree of coincidence of spectra is disclosed in detail in, for example, Patent Document 1.

  [Method 2] The similarity between the absorption spectrum of the peak top and each absorption spectrum from the peak start time to the peak end time and a threshold are calculated, and the value of [similarity]-[threshold] is calculated with respect to the time axis. Some plots determine the purity of a peak by determining whether the value of [similarity]-[threshold] may be negative during the time the peak appears (non-saturation). Patent Document 2).

  However, in the above method 1, the calculation can be performed relatively easily. However, since the entire profile of the elution peak is not examined closely, for example, when an impurity is overlapped near the end of the peak, it cannot be detected. There is a risk of misjudging that it is one peak. In addition, a threshold value for determining the average value of the degree of coincidence needs to be given in advance, but the certainty cannot be guaranteed statistically.

  On the other hand, in method 2, it is necessary to set the noise level as a condition in order to determine the threshold value, but in the PDA detector, the noise level differs for each wavelength. In addition, there is no guarantee that the noise level (uncertainty) on the baseline is equal to the uncertainty at a portion where the signal strength is high. For this reason, in order to accurately determine the peak purity by the method 2, it is inevitable that the process is inevitably complicated.

  In addition, in any method, it is only possible to determine whether the target peak is due to a single component or not, and it is possible to estimate how many components may overlap if they are not single components. Can not do.

Japanese Patent Publication No. 7-74761

Yasutaka Mito, Mitsuo Kitaoka, “Photodiode Array UV-VIS Detector SPD-M6A for Shimadzu HPLC”, Shimazu Review, Vol. 46, No. 1, July 1989, p. 21-28 "LC workstation CLASS-VP Ver6.1 Instruction Manual", Shimadzu Corporation, April 2001, p. 9-40 to 9-41

  The present invention has been made in view of the above problems, and its main purpose is to make it possible to perform highly accurate peak purity determination with a small amount of calculation without the user performing complicated setting of conditions and the like. The object is to provide a chromatographic data processing apparatus.

In order to solve the above-described problems, the present invention provides a chromatograph for processing three-dimensional data with dimensions of time, channel, and signal intensity collected by a chromatograph using a multi-channel detector as a detector. A chromatograph data processing apparatus for determining the purity of a peak present in a chromatogram created based on the data,
a) data extraction means for extracting three-dimensional data of a plurality of channels all or part of the time range from the start point to the end point of the target peak;
b) Create a matrix using the data extracted by the data extraction means as matrix elements, perform principal component analysis on the matrix, and from the first principal component to the n-th principal component (n is an integer of 2 or more) A principal component analysis means for calculating the variance of
c) contribution rate calculating means for calculating the contribution rate of the first principal component or the cumulative contribution rate after the first principal component from the variance of each principal component obtained by the principal component analysis;
It is characterized by having.

  In the chromatographic data processing apparatus according to the present invention, the multi-channel detector is, for example, a photodiode array (PDA) detector or a wavelength scanning ultraviolet-visible spectroscopic detector, and in this case, the channel is a wavelength. It is. A mass spectrometer can also be used as a multi-channel detector. In this case, the channel is a mass-to-charge ratio (m / z value). In particular, since a PDA detector can detect multiple wavelengths simultaneously, absorption spectrum data can be repeatedly acquired at narrow time intervals.

  The chromatograph may be either a liquid chromatograph or a gas chromatograph.

  In addition, the start time point and the end time point of the target peak may be specified by the user by operating the mouse on the chromatogram drawn on the display screen, for example, or detected as a result of the peak detection process. The peak start time and end time may be automatically set. That is, the target peak and its start / end time may be set manually or automatically.

  In any case, when the start time point and end time point of the target peak to be subjected to peak purity determination, that is, the time range of the peak is determined, the data extraction means, for example, from the storage unit storing the data in the corresponding time range Read 3D data. The data used at this time is preferably all (that is, not thinned out) data on both the time axis and the channel axis, but is not necessarily limited to this, and for example, data of some of a plurality of channels may be used.

  The principal component analysis means creates a matrix having the extracted data as matrix elements, and performs principal component analysis on the matrix. In other words, if the extracted data is N on the time axis and M on the channel axis, a matrix with M variables and N samples is created, and the principal component for this matrix is created. Perform analysis. By the principal component analysis, respective variances from the first principal component to the n-th principal component are obtained. Although the maximum value of n is determined by the number of variables, it is not always necessary to obtain the maximum value, and can be cut off by an appropriate number. Each main component corresponds to a sample component included in the target peak. Further, it can be said that the larger the variance value, the more the original data can be explained by the principal component.

  Therefore, the contribution rate calculation means first calculates the contribution rate of the first principal component from the variance of each principal component obtained by the principal component analysis. If the contribution ratio of the first principal component is sufficiently high, the original data can be explained only by the first principal component. That is, this means that one sample component is included in the target peak. On the other hand, when the contribution rate of the first principal component is not sufficiently high, the contribution rate calculation means sequentially calculates the cumulative contribution rate up to and after the second principal component. For example, when the cumulative contribution ratio up to the second principal component is sufficiently high, it can be said that the original data can be explained by two of the first principal component and the second principal component that are not correlated with each other. That is, this means that there are two sample components included in the target peak. By repeating this until the cumulative contribution ratio shows a sufficiently high value, it is possible to estimate that the sample component included in the target peak is 3 or more.

  In the chromatographic data processing apparatus according to the present invention, preferably, the purity estimation means for estimating the number of components overlapping the target peak based on the contribution rate or the cumulative contribution rate calculated by the contribution rate calculation means, It is good to have a configuration provided.

  The purity estimation means compares the calculated contribution rate or cumulative contribution rate with a predetermined threshold value, and if the contribution rate or cumulative contribution rate is equal to or greater than the threshold value, the purity estimation means The number may be estimated to be the number of sample components. The threshold value may be determined in advance as 0.8 (= 80%), for example, or may be set appropriately by the user.

  The chromatographic data processing apparatus according to the present invention further comprises a graph creating means for creating a graph showing the relationship between each principal component obtained by principal component analysis and the dispersion and rendering it on the display screen. You can also

  When the target peak is due to a single sample component, that is, when the target peak is completely pure, only the dispersion of the first principal component is prominently large, and the dispersion of the second and subsequent principal components is all small. Become. Further, when there are two sample components included in the target peak, the dispersion of the first principal component and the second principal component is prominently increased, and the dispersion of the third and subsequent principal components is all reduced. Therefore, if a graph showing the relationship between each principal component and the dispersion is displayed, the user can visually recognize the purity of the peak (overlap of sample components) immediately by looking at this graph. In addition, it is possible to confirm the appropriateness of the determination based on the contribution rate and the cumulative contribution rate using this graph.

  According to the chromatographic data processing apparatus of the present invention, the purity of the peak can be determined using all or most of the three-dimensional data corresponding to the peak appearing in the chromatogram. Therefore, for example, even when other sample components overlap only in the vicinity of the end of the peak, the overlap can be detected accurately, and the accuracy of peak purity determination can be improved.

  Further, in the chromatographic data processing apparatus according to the present invention, it is not necessary to set complicated conditions and parameters for determining the peak purity, and the calculation itself is not so complicated. Therefore, an excessive burden is not imposed on the user for setting the conditions, and the peak purity determination can be performed efficiently, that is, with a high throughput.

  Furthermore, the chromatographic data processing apparatus according to the present invention not only determines that a peak is not a single component, but also can estimate the number of overlapping sample components with high accuracy. Information more useful than processing can be provided to the user.

The block diagram of the principal part of the liquid chromatograph which is one Example of the chromatograph using the data processor which concerns on this invention. The conceptual diagram of the three-dimensional data and chromatogram obtained with the liquid chromatograph of a present Example. The flowchart which shows the peak purity determination processing procedure in the liquid chromatograph of a present Example. The figure which shows an example of the main component-dispersion value graph displayed with the liquid chromatograph of a present Example.

  Hereinafter, a liquid chromatograph which is an embodiment of a chromatograph using a data processing apparatus according to the present invention will be described with reference to the accompanying drawings.

  FIG. 1 is a configuration diagram of a main part of the liquid chromatograph of the present embodiment. In this liquid chromatograph, the liquid feed pump 2 sucks the mobile phase accommodated in the mobile phase container 1 and feeds it to the column 4 through the injector 3 at a substantially constant flow rate. The injector 3 injects a sample prepared in advance into the mobile phase at a predetermined timing. The injected sample liquid is introduced into the column 4 along the flow of the mobile phase, and various sample components contained in the sample liquid are separated while passing through the column 4 and are eluted from the column 4 with time lag.

  A PDA detector 5 is provided at the outlet of the column 4 as a multi-channel detector that detects a sample component in the eluate from the column 4. The PDA detector 5 includes a transparent flow cell 51 through which an eluate flows, a light source 52, a spectroscope 53 that wavelength-disperses light transmitted through the flow cell 51, and a PDA element 54 that detects wavelength-dispersed light almost simultaneously. ,including.

  The detection signal from the PDA detector 5 is converted into a digital signal by the A / D converter 6 and input to the data processing unit 7 and temporarily stored in the data memory 8. Thereafter, the data processing unit 7 executes various processes as will be described later on the data stored in the data memory 8. In particular, the data processing unit 7 includes a data extraction unit 71, a principal component analysis unit 72, a contribution rate calculation unit 73, a component number determination unit 74, and a principal component-dispersion as functional blocks for performing a peak purity determination process described later. A graph display processing unit 75 is provided.

  The analysis control unit 9 controls each unit such as the liquid feeding pump 2 based on an instruction from the control unit 10. The control unit 10 is connected to an operation unit 11 such as a keyboard and a mouse operated by a person in charge of analysis, and a display unit 12 for displaying measurement results, and is mainly related to such a user interface. Note that most or some of the functions of the analysis control unit 9, the control unit 10, and the data processing unit 7 are performed by using a general-purpose personal computer as hardware and operating dedicated control / processing software installed in advance on the hardware. It can be set as the structure implement | achieved by.

  Since the PDA detector 5 can detect multiple wavelengths simultaneously without performing wavelength scanning, the absorption spectrum data in a predetermined wavelength range is acquired at predetermined time intervals from the time when the sample is injected into the mobile phase by the injector 3. Can do. Therefore, in this liquid chromatograph, three-dimensional data having three dimensions of time, wavelength, and signal intensity as shown in FIG. When a liquid chromatographic analysis is performed on a certain sample under the control of the analysis control unit 9, three-dimensional data from the analysis start time (sample injection time) to the analysis end time is stored in the data memory 8. .

  Next, characteristic peak purity determination processing that is executed mainly in the data processing unit 7 in the liquid chromatograph of the present embodiment will be described with reference to FIGS. 3 and 4. FIG. 3 is a flowchart showing the procedure of the peak purity determination process.

  When the person in charge of analysis performs a predetermined operation with the operation unit 11, the data processing unit 7 renders a chromatogram of a specific wavelength on the screen of the display unit 12. The wavelength at that time can be arbitrarily specified. The person in charge of the analysis instructs the peak start time ts and the peak end time te of the target peak for which the peak purity determination is to be performed using the operation unit 11 while viewing the chromatogram (step S1). FIG. 2B is a diagram illustrating an example of a target peak, a peak start time ts, and a peak end time te.

  Instead of manually specifying the target peak start time and end time by the analyst (user) himself, when the user specifies the target peak, the peak start time and end time are automatically obtained. You may do it. Alternatively, the peak detection itself may be automatically executed, and the start point and the end point may be automatically set for all detected peaks. Such automatic peak detection and peak start point and end point detection can be performed using a conventional peak detection method.

この行列Ｘの要素ｘijは、時間軸上でｉ、波長軸上でｊの位置にあるデータ値、つまり信号強度値である。 In the data processing unit 7, the data extraction unit 71 extracts all three-dimensional data corresponding to the designated peak start time ts to peak end time te from the three-dimensional data stored in the data memory 8. Read (step S2). Hereinafter, a large number of the read three-dimensional data in the time range from ts to te are collectively referred to as an extracted data group. Assuming that the number of data included in the extracted data group is N in the time axis direction and the number of wavelength points in the wavelength axis direction is M, the entire extracted data group can be represented by the following matrix.

The element xij of the matrix X is a data value at a position i on the time axis and j on the wavelength axis, that is, a signal intensity value.

  The matrix is a matrix having N variables and M samples. The principal component analysis unit 72 performs principal component analysis on this matrix and calculates variances of the first to nth principal components (step S3). The number of sample data for obtaining the principal component only needs to be larger than the vector order defining the sample data. In principal component analysis, principal components having the same number of vector dimensions as the sample data space are obtained. The principal component that determines the axis with the largest variance of sample data is the first principal component, and the axis with the second largest variance is determined. The main component is the second main component, and the nth main component is determined in the same manner.

  When the variance from the first principal component to the n-th principal component is found, the principal component-dispersion graph display processing unit 75 uses the principal component # as the horizontal axis and the variance value as the vertical axis, and sets the variance value of each principal component to the first. A graph in which the principal components are arranged and plotted in order is created, and this is drawn on the display unit 12 via the control unit 10 (step S4). If the chromatogram peak to be determined is derived from a single sample component, the significant principal component is only the first principal component, so that only the dispersion value of the first principal component is the variance of the second and subsequent principal components. Prominently larger for the value. In addition, the dispersion values after the second main component are almost equal.

  On the other hand, the contribution ratio calculation unit 73 first calculates the contribution ratio of the first principal component using the respective variance values of the first principal component to the n-th principal component (step S5). And the component number determination part 74 determines whether the calculated contribution rate is 0.8 or more which is a threshold value (step S6), and if it is 0.8 or more, the peak is pure. That is, it is determined that the sample is composed of only one sample component (step S7), and a display notifying that is given to the display unit 12 (step S12).

  When the contribution rate of only the first principal component is less than 0.8, the contribution rate calculation unit 73 calculates the cumulative contribution rate up to the second principal component (step S8), and this cumulative contribution rate is 0.8. It is determined whether or not this is the case (step S9). If the cumulative contribution rate up to the second main component is 0.8 or more, it is determined that the peak is an elution of the two components overlapping (step S10), and a display to that effect is displayed on the display unit 12. It performs (step S12). If the cumulative contribution rate up to the second principal component is less than 0.8, the cumulative contribution rate up to and after the third principal component is further calculated and the determination for the threshold is executed in the same manner to obtain the number of overlapping components (step) S11).

  Thereby, on the screen of the display part 12, the graph which shows the relationship between a main component and a dispersion | distribution value, and the number of components which have overlapped with the target peak estimated based on this relationship are displayed. FIG. 4A is an example of a graph when the peak is composed of a single component, and FIG. 4B is an example of a graph when the peak is composed of two components.

  In addition, the threshold value for determining the contribution rate or the cumulative contribution rate can be changed as appropriate, and for example, the user may freely set it from the operation unit 11 or the like.

  In the above embodiment, the three-dimensional data in the predetermined time range corresponding to the target peak is cut out from all the collected three-dimensional data, but it is not always necessary to use the data of all the wavelength points, and the partial wavelength Data included in the range or data for a plurality of distant wavelengths may be used. Practically, it is better to use a larger number of wavelengths, but theoretically, the above processing can be performed by using chromatogram data in a predetermined time range at a minimum of two wavelengths.

  In the above embodiment, the data processing apparatus according to the present invention is applied to a liquid chromatograph using a PDA detector, but instead of the PDA detector, wavelength scanning is performed to collect spectrum data in a predetermined wavelength range. Needless to say, the present invention can be applied to a liquid chromatograph using an ultraviolet-visible spectroscopic detector.

  Further, the collected data is not data having three dimensions of time, wavelength, and signal intensity, but may be data having another dimension instead of the wavelength. For example, a liquid chromatograph mass spectrometer or gas chromatograph mass spectrometer using a mass spectrometer as a detector can obtain data having three dimensions of time, mass-to-charge ratio, and signal intensity. You can also

DESCRIPTION OF SYMBOLS 1 ... Mobile phase container 2 ... Liquid feed pump 3 ... Injector 4 ... Column 5 ... PDA detector 51 ... Flow cell 52 ... Light source 53 ... Spectroscope 54 ... PDA element 6 ... A / D converter 7 ... Data processing part 71 ... Data Extraction unit 72 ... principal component analysis unit 73 ... contribution rate calculation unit 74 ... component number determination unit 75 ... principal component-dispersion graph display processing unit 8 ... data memory 9 ... analysis control unit 10 ... control unit 11 ... operation unit 12 ... display Part

Claims (4)

A chromatographic data processing apparatus for processing three-dimensional data with dimensions of time, channel, and signal intensity collected by a chromatograph using a multi-channel detector as a detector, and created based on the data In the chromatographic data processing apparatus for determining the purity of the peak present in the chromatogram to be performed,
a) data extraction means for extracting three-dimensional data of a plurality of channels all or part of the time range from the start point to the end point of the target peak;
b) Create a matrix using the data extracted by the data extraction means as matrix elements, perform principal component analysis on the matrix, and from the first principal component to the n-th principal component (n is an integer of 2 or more) A principal component analysis means for calculating the variance of
c) contribution rate calculating means for calculating the contribution rate of the first principal component or the cumulative contribution rate after the first principal component from the variance of each principal component obtained by the principal component analysis;
A chromatograph data processing apparatus comprising: The chromatographic data processing apparatus according to claim 1,
A chromatographic data processing apparatus, further comprising: a purity estimating unit that estimates the number of components overlapping with a target peak based on a contribution rate or a cumulative contribution rate calculated by the contribution rate calculating unit. A chromatographic data processing apparatus according to claim 1 or 2,
A chromatographic data processing apparatus, further comprising: a graph creating unit that creates a graph showing a relationship between each principal component obtained by the principal component analysis and the variance and displays the graph on a display screen. A chromatographic data processing apparatus according to any one of claims 1 to 3,
The chromatograph data processing apparatus, wherein the multi-channel detector is a photodiode array detector, and the channel is a wavelength.

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