JP2011185743A - Chromatogram peak purity determination apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chromatogram peak purity determination apparatus for accurately determining a peak purity at a high concentration. <P>SOLUTION: The three-dimensional chromatogram peak purity determination apparatus for a retention time, a wavelength and the absorbance comprises: an absorbance accumulation means 71 for accumulating the absorbance in the entire or partial wavelength range for a predetermined retention time range at a retention time in the wavelength direction; an equal absorbance accumulation value group extracting means 73 for extracting a plurality of groups including one point on a rising slope and one point on a falling slope on which absorbance accumulation values are equal with regard to a peak of a two-dimensional chromatogram of the absorbance accumulation value and the retention time; a similarity calculation means 74 for calculating the similarity between absorption spectrum at one point on the rising slope and absorption spectrum at one point on the falling slope in each extracted group; and a determination means 76 for determining whether the peak is a single component from the similarity calculated by a plurality of the extracted groups. Since the spectrum similarity between the high concentration and the low concentration is not compared, an effect for degrading the linearity at the high concentration does not impact. The peak purity can be accurately determined at the high concentration. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a data processing apparatus for processing data obtained from a liquid chromatograph or the like, and more specifically, a peak appearing in a chromatogram obtained from a chromatograph using a multichannel detector such as a photodiode array detector. The present invention relates to an apparatus for determining the purity.

  When a sample is analyzed by a liquid chromatograph or the like, a peak corresponding to a component contained in the analyzed sample appears on the chromatogram. However, the peak that appears to be a single shape on 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 of the liquid chromatograph overlap. Therefore, conventionally, a chromatogram peak purity determination device has been used to determine whether a peak on a chromatogram is due to a single component.

  Non-Patent Document 1 discloses a peak purity determination method in a chromatogram obtained by a liquid chromatograph using a photodiode array (PDA) detector. With a PDA detector, data is obtained that includes three dimensions: wavelength, retention time, and signal strength. The above document describes two methods for determining peak purity.

  In the first method, when determining the purity of a certain peak on the chromatogram, the absorption spectrum patterns at the three points of the peak top, the peak rising slope, and the peak falling slope are compared, and the spectrum at the peak top is compared. The degree of coincidence between the pattern and the spectrum pattern in the peak rising slope and the degree of coincidence between the spectrum pattern in the peak top and the spectrum pattern in the peak falling slope are respectively obtained. (The method for obtaining the degree of coincidence of spectral patterns is disclosed in detail in Patent Document 1.) Then, an average value of the two coincidences is obtained, and it is simply determined whether or not it is larger than a predetermined threshold value. It is determined whether or not there is one peak.

  Another method is to calculate the similarity and threshold between the absorption spectrum of the peak top and each absorption spectrum from the peak start time to the peak end time, and the value of [similarity]-[threshold] is calculated on the time axis. And the purity of the peak is determined by determining whether or not the value of [similarity]-[threshold] may be negative during the time when the peak appears. (Non-patent document 2).

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, pp. 21-28 “LC Workstation CLASS-VP Ver6.1 Instruction Manual”, Shimadzu Corporation, April 2001, pp. 9-40 to 9-41

  In an absorptiometer detector of a liquid chromatograph, the linearity of concentration versus absorbance decreases in a high concentration region due to the influence of stray light. For this reason, even if it is the same component, a spectrum pattern in a low concentration spectrum and a high concentration spectrum do not completely coincide. Therefore, in the above two methods, when the peak top has a high concentration, there is a risk that the purity is judged to be low even if the peak contains only a single component. In addition, in the low wavelength region (190 nm to 220 nm) where the energy of the light source lamp is low, the influence of the decrease in linearity is particularly significant, resulting in spectral distortion. Therefore, also in this case, the purity is judged to be low even for a peak composed of a single component.

  This invention is made | formed in view of said problem, and makes it a subject to provide the chromatogram peak purity determination apparatus which can maintain the precision of peak purity determination also in a high concentration area | region.

The chromatogram peak purity determination device according to the first aspect of the present invention, which has been made to solve the above-described problems, is collected by a chromatograph using a photodiode array detector as a detector, a retention time, a wavelength, and In a chromatograph data processing apparatus for processing three-dimensional data having a dimension of absorbance, a chromatogram peak purity determination apparatus for determining peak purity existing in a chromatogram created based on the three-dimensional data,
a) Absorbance integration means for integrating the absorbance of all or part of the wavelength range in the predetermined holding time range in the wavelength direction for each holding time;
b) Concerning the peak existing in the chromatogram created based on the two-dimensional data with the absorbance integrated value and the retention time as a dimension, the absorbance integrated value consists of one point on the rising slope and one point on the falling slope. An equal absorbance integrated value group extraction means for extracting a plurality of sets, and
c) Similarity calculation for calculating the similarity between the absorption spectrum pattern at one point on the rising slope and the absorption spectrum pattern at one point on the falling slope for each set extracted by the iso-absorbance integrated value group extraction means Means,
d) determination means for determining whether or not the peak is a single component based on the similarity calculated by the similarity calculation means for the plurality of sets extracted by the absorbance integrated value set extraction means;
It is characterized by providing.

  Here, the chromatograph may be either a liquid chromatograph or a gas chromatograph. The “predetermined retention time range” refers to a preset time range of the absorbance detection time, and it is sufficient that at least the time range from the start to the end of the peak is included.

The chromatogram peak purity determination apparatus according to the second aspect of the present invention is the chromatogram peak purity determination apparatus according to the first aspect,
The absorbance integration means is characterized in that the absorbance in all or a part of the wavelength range in the holding time range from the start time to the end time of the peak of the three-dimensional chromatogram is integrated in the wavelength direction for each holding time.

  The chromatogram peak purity determination device according to the third aspect of the present invention is the chromatogram peak purity determination device according to the first or second aspect, wherein the similarity calculation means absorbs one point on the rising slope. The spectral pattern and the absorption spectral pattern at one point on the descending slope are each represented by a vector having the absorbance for each wavelength as an element, and the narrow cosine formed by these vectors is calculated as the similarity.

The chromatogram peak purity determination apparatus according to the fourth aspect of the present invention is the chromatogram peak purity determination apparatus according to the third aspect,
The determination means is characterized in that determination is made by comparing an average value of a plurality of sets of cosines with a determination value.

The chromatogram peak purity determination apparatus according to the fifth aspect of the present invention is the chromatogram peak purity determination apparatus according to the third aspect,
The determination means is characterized by determining by comparing a plurality of sets of cosine variances with a determination value.

The chromatogram peak purity determination method according to the sixth aspect of the present invention,
Created based on the three-dimensional data in a chromatographic data processing device that processes three-dimensional data collected using a photodiode array detector as a detector and having dimensions of retention time, wavelength, and absorbance. A chromatogram peak purity determination method for determining peak purity present in a chromatogram,
Absorbance integration step of integrating the absorbance of all or part of the wavelength range in a predetermined holding time range in the wavelength direction for each holding time;
For a peak present in a chromatogram created on the basis of the absorbance integrated value integrated in the absorbance integrating step and the two-dimensional data having the retention time as a dimension, one point on the rising slope and the falling slope are equal to each other. Iso-absorbance integrated value set extraction step for extracting a plurality of sets consisting of one point of
A similarity calculation step for calculating the similarity between the absorption spectrum pattern at one point on the rising slope and the absorption spectrum pattern at one point on the falling slope for each set extracted in the equal absorbance integrated value group extraction step; ,
A determination step for determining whether or not the peak is a single component based on the similarity calculated in the similarity calculation step for a plurality of sets extracted in the equiabsorbance integrated value group extraction step;
It is characterized by including.

  In the chromatogram peak purity determination apparatus according to the present invention, in a two-dimensional chromatogram in which the absorbance is integrated in the wavelength direction for each holding time and the calculated absorbance integrated value and the holding time are dimensions, spectra having the same absorbance integrated value are used. Plural sets are detected, and the purity of the peak is determined by calculating the similarity of the spectral pattern for each set.

  For the calculation of the similarity between the spectral patterns, the method described in Patent Document 1 is referred to. Patent Document 1 discloses a concept of calculating a similarity from the direction of a vector by representing an absorption spectrum as a set of discrete absorbance data detected in a series of wavelength sequences and treating it as a vector.

Specifically, the absorption spectrum is expressed as S → = (a (λ1), a (λ2), a (λ3) .., a (λn)). Here, what is indicated with → indicates a vector. Further, a (λn) indicates the absorbance at the wavelength λn.
If each of the two absorption spectra is represented as a vector as described above, if these spectra are composed of exactly the same components, the spectrum patterns are equal, and the vectorized spectra are directed in the same direction. Here, if the similarity is defined as the cosine value of the narrow angle θ formed by the two vectorized spectra, if the two vectors are exactly the same, θ is 0 and the cosine value cos θ is 1. That is, the closer the similarity is to 1, the more similar the spectral pattern.

  In the present invention, based on the above idea, for a single peak, a plurality of spectrum sets having the same absorbance integrated value are extracted from the rising slope and the falling slope, and the degree of similarity is examined by vectorizing the spectra for each set. .

によりKが計算される。 The determination of the peak purity based on the similarity is performed by comparing the average value of the similarity with the determination value. Here, the determination value (K) is the maximum allowable value for determining that the spectrum patterns are similar in consideration of the influence of detection noise and the like of the detector. It is assumed that the originally identical vector has shifted by n → due to the influence of noise, and S1 → is displaced by θ1 from the true vector S1 ′ →, and S2 → is displaced by θ2 from the true vector S2 ′ → (FIG. 1). The coincidence between the true vectors S1 ′ → and S2 ′ → is realized in a range where the narrow angle between S1 → and S2 → is the maximum θ1 + θ2. Therefore, the determination value (K), which is the maximum allowable range of vector similarity, is cos (θ1 + θ2). Where Equation 1

Calculates K.

  Thus, in the chromatogram peak purity determination apparatus according to the present invention, the purity of the peak is determined by comparing the similarity of the spectrum patterns with the spectrum set having the same integrated absorbance value. In a set of equal values obtained by integrating the absorbance in the wavelength direction, it is estimated that the concentrations of the samples detected in the respective holding times are almost equal. Therefore, in the present invention, the degree of similarity is compared between spectrum sets having substantially the same concentration. In this way, since the similarity of spectra is not compared between high and low concentrations, the accuracy of peak purity determination can be maintained even at high concentrations without being affected by the decrease in linearity at high concentrations. It becomes possible.

Explanatory drawing explaining the spectrum pattern similarity determination method in this invention. The principal part block diagram of the liquid chromatograph which is one Example of the chromatograph using the chromatogram peak purity determination apparatus which concerns on this invention. (a) is a graph of three-dimensional data obtained by the liquid chromatograph of this example, (b) is a chromatogram in which the integrated absorbance value in the wavelength direction is plotted in a predetermined time range in the time direction, and (c) is the wavelength. The chromatogram which plotted the absorbance integrated value of the direction from the peak start to the end in the time direction is shown. Explanatory drawing explaining the method to divide | segment the peak of the chromatogram examined in the present Example. The spectrum figure which plotted the spectrum in the peak of the chromatogram examined in the present Example in the wavelength direction.

  A liquid chromatograph which is an embodiment of a chromatograph using the peak purity determination apparatus according to the present invention will be described with reference to FIGS.

  FIG. 2 shows a main configuration of a liquid chromatograph using the chromatogram peak purity determination apparatus according to 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 that detects sample components in the eluate from the column 4 is provided at the outlet of 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 functions as a functional block for performing peak purity determination processing described later, an absorbance integration unit 71, a chromatogram generation unit 72, a spectrum set detection unit 73, a similarity calculation unit 74, and a similarity statistic value calculation. Unit 75 and peak purity determination unit 76.

  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 the user, and a display unit 12 for displaying measurement results and the like, and 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 Ti, wavelength λj, and signal intensity Aij 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.

Prior to sample analysis with a liquid chromatograph, the user sets the range of the wavelength and holding time in which the peak of the sample is expected to be detected from the operation unit 11. When the analysis is completed, the absorbance integrating unit 71 reads out the three-dimensional data from the data memory 8 in accordance with a user instruction from the operation unit 11, and sets a predetermined wavelength range within a preset holding time range (Tp to Tq). The absorbance of (λr to λs) is integrated for each holding time. Here, the absorbance integration is
Atotal (i) = ΣA (i, j) (j = r〜s)
Do by.

  In this embodiment, the wavelength and holding time range is preset as the absorbance integration range, but the holding time range and wavelength range in which a three-dimensional chromatogram is drawn on the display screen and the user integrates with a mouse operation or the like. May be specified.

  The chromatogram generation unit 72 renders a two-dimensional chromatogram on the screen of the display unit 12 in which the absorbance integrated value Atotal (i) calculated by the absorbance integrating unit 71 is plotted in the direction of holding time Ti (i = p to q). (FIG. 3B).

  The spectrum set detection unit 73 equally divides the peak top height in the two-dimensional chromatogram generated in the chromatogram generation unit 72 into a plurality of points, and sets one point on the rising slope with the same integrated absorbance value. Find the equiabsorbance set at one point on the descending slope. In this example, the peak top height is divided into seven equal parts to obtain six sets of equal absorbance integrated values (see FIG. 4). Here, Su1 → ˜Su6 → and Sd1 → ˜Sd6 → indicate the spectrum at each point as a vector, for example, Su1 → = (A (λr)... A (λs)).

Next, the similarity calculation unit 74 calculates the spectrum similarity R for each equal absorbance set detected by the spectrum set detection unit 73. The degree of similarity is defined by the cosine value cos θ of the narrow angle θ formed by vectorizing the spectrum at each point on the rising slope of the peak and the corresponding point on the falling slope. Specifically, it is calculated by the following (Formula 2).

である。ここで、n→はノイズによるずれをベクトル化したものである。 The similarity statistic value calculation unit 75 calculates the average value R _ave of the similarity in all sets using the spectrum similarity R of each equal absorbance set calculated by the similarity calculation unit 74. In addition, a determination value that is the maximum allowable range of similarity for determining that the spectra are the same is also calculated. Here, the judgment value K is

It is. Here, n → is a vectorized shift due to noise.

The peak purity determination unit 76 determines whether or not the designated peak is composed of a single component from the average value of the similarity calculated by the similarity statistical value calculation unit 75 and the determination value. Specifically, the peak purity determination unit 76 compares the average value R _ave of the similarity R calculated by the similarity statistical value calculation unit 75 with the average value K _ave of the determination value K,
R _ave ≧ K _ave
In this case, it is determined that the spectra of the equiabsorbance set are the same. The fact that the spectra of isoabsorbance groups are the same means that the rising slope and the falling slope of the peak are the same, and that the peak is composed of a single component.

The present invention has been made in view of the fact that the linearity of concentration versus absorbance decreases in a high concentration region. Here, the influence on the spectrum pattern due to the difference in concentration was examined.
FIG. 5 shows a spectrum obtained by plotting the spectra Su1, Su4, Su6, Sd6, Sd4, and Sd1 in the wavelength direction for the peaks examined in this example.

  From FIG. 5, it can be seen that the spectral patterns are greatly different in low concentration Su1, Sd1, high concentration region Su6, Sd6, and intermediate concentration regions Su4, Sd4. On the other hand, the spectrum patterns of Su1 and Sd1, Su4 and Sd4, and Su6 and Sd6 are completely identical. As described above, the influence of the concentration on the spectrum pattern is considerably large. On the other hand, since the spectrum pattern completely matches in the region where the concentration is equal, the chromatogram peak purity determination device according to the present invention is higher than the conventional determination device. It is considered that peak purity can be determined with high accuracy.

  In addition, this invention is not limited to the said Example, A change is accept | permitted within the range of the meaning of invention. For example, in the above embodiment, the absorbance is integrated in a preset holding time range, but the absorbance may be integrated from the start to the end of the target peak (see FIG. 3 (c)). The absorbance may be integrated from before the peak start time to after the peak end time. When integrating from the peak start to the end, the operation unit 11 instructs the user to start and end the target peak for which peak purity determination is desired. Instead of manually specifying the peak start time and end time by the user himself / herself, when the user specifies the target peak, the start time and end time of the peak may be automatically obtained. 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 / end detection can be performed using a conventional peak detection method.

  In the above embodiment, the absorbances detected at all wavelengths in the range of λr to λs set in advance are integrated, but only the absorbances detected at a plurality of specific wavelengths may be integrated (in short, thinning out). In extreme cases, it may be configured to integrate the absorbance at two wavelengths.

  In this embodiment, when detecting multiple sets of integrated absorbance values from a single peak on the chromatogram, the peak top height is equally divided into seven, but the peak top height is equally divided and set. The number of divisions is not limited to seven. Further, it is not necessary to create a two-dimensional chromatogram using the integrated value in order to detect the equiabsorbance integrated value set, and the isoabsorbed integrated value set can be detected only by calculation.

In this embodiment, the peak purity is determined by comparing the determination value average value K _ave obtained by calculation with the similarity average value R _ave , but the determination value may be set based on an experiment.

  In this embodiment, the average value of similarity is used as a basis for determination of peak purity. However, it is also possible to determine peak purity by using similarity variance and standard deviation. Similarity variance and standard deviation are performed by the similarity statistical value calculator 75. When the peak is composed of a single component, the similarity R (= cos θ) of the iso-absorbance group on the rising slope and the falling slope is close to 1. Accordingly, the variance and standard deviation of the similarity in all iso-absorbance groups are small values. The peak purity determination unit 76 determines the purity of the peak by comparing with a threshold value set in advance by the user.

  As a modification, instead of integrating the absorbance in a predetermined wavelength range, a two-dimensional chromatogram in which the signal intensity at a specific wavelength is plotted in the time direction is generated, and an iso-absorbance set is extracted from the chromatogram. Can also be considered.

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 ... Absorbance integration unit 72 ... chromatogram generation unit 73 ... spectrum group detection unit 74 ... similarity calculation unit 75 ... similarity statistical value calculation unit 76 ... peak purity determination unit 8 ... data memory 9 ... analysis control unit 10 ... control unit 11 ... Operation unit 12 ... Display unit

Claims (6)

Created based on the three-dimensional data in a chromatographic data processing device that processes three-dimensional data collected using a photodiode array detector as a detector and having dimensions of retention time, wavelength, and absorbance. A chromatogram peak purity determination device for determining peak purity existing in a chromatogram,
a) Absorbance integration means for integrating the absorbance of all or part of the wavelength range in the predetermined holding time range in the wavelength direction for each holding time;
b) Concerning the peak existing in the chromatogram created based on the two-dimensional data with the absorbance integrated value and the retention time as a dimension, the absorbance integrated value consists of one point on the rising slope and one point on the falling slope. An equal absorbance integrated value group extraction means for extracting a plurality of sets, and
c) Similarity calculation for calculating the similarity between the absorption spectrum pattern at one point on the rising slope and the absorption spectrum pattern at one point on the falling slope for each set extracted by the iso-absorbance integrated value group extraction means Means,
d) determination means for determining whether or not the peak is a single component based on the similarity calculated by the similarity calculation means for the plurality of sets extracted by the absorbance integrated value set extraction means;
A chromatogram peak purity determination apparatus comprising: In the chromatogram peak purity determination device according to claim 1,
The absorbance integrating means integrates the absorbance in all or a part of the wavelength range in the holding time range from the start time to the end time of the peak of the three-dimensional chromatogram in the wavelength direction for each holding time. Gram peak purity determination device. In the chromatogram peak purity determination apparatus according to claim 1 or 2,
The similarity calculation means represents an absorption spectrum pattern at one point on the rising slope and an absorption spectrum pattern at one point on the falling slope by vectors each having the absorbance for each wavelength as an element, and a narrow cosine formed by these vectors. As a similarity, a chromatogram peak purity determination device. In the chromatogram peak purity determination apparatus according to claim 3,
The determination means is a chromatogram peak purity determination device characterized in that determination is made by comparing an average value of a plurality of sets of cosines with a determination value. In the chromatogram peak purity determination apparatus according to claim 3,
The determination means is a chromatogram peak purity determination apparatus characterized in that a determination is made by comparing a plurality of sets of cosine variances with a determination value. Created based on the three-dimensional data in a chromatographic data processing device that processes three-dimensional data collected using a photodiode array detector as a detector and having dimensions of retention time, wavelength, and absorbance. A chromatogram peak purity determination method for determining peak purity present in a chromatogram,
Absorbance integration step of integrating the absorbance of all or part of the wavelength range in a predetermined holding time range in the wavelength direction for each holding time;
For a peak present in a chromatogram created on the basis of the absorbance integrated value integrated in the absorbance integrating step and the two-dimensional data having the retention time as a dimension, one point on the rising slope and the falling slope are equal to each other. Iso-absorbance integrated value set extraction step for extracting a plurality of sets consisting of one point of
A similarity calculation step for calculating the similarity between the absorption spectrum pattern at one point on the rising slope and the absorption spectrum pattern at one point on the falling slope for each set extracted in the equal absorbance integrated value group extraction step; ,
A determination step for determining whether or not the peak is a single component based on the similarity calculated in the similarity calculation step for a plurality of sets extracted in the equiabsorbance integrated value group extraction step;
A method for determining chromatogram peak purity, comprising:

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