JPH07103959A - Chromatogram analysis method, chromatograph device and data processor used for them - Google Patents

Abstract

PURPOSE:To exactly find a peak area and contrive the improvement of analysis precision by deconvolution-processing detection peak waveforms by means of a spreading function, correcting waveform strain of chromatogram, sharpening the overlapped peak waveforms and separating them. CONSTITUTION:A standard sample 52 wherein a non-holding component is added is infected, a peak which is larger than size which appears at the beginning time of chromatogram is automatically identified as a non-holding wave peak or time designation is performed from an input part 54, a mouse non- holding wave peak is extracted and designated from the chromatogram shown in CRT 46, its area is normalized and a normalization function (spreading function) is found. Next, the start point and the end point of a time of the overlapped peak 64, 64 desired to be measured are set, convolution processing is performed in the time interval by means of the normalization function, integration intended for the peak waveform after sharpened processing is conducted, and measurement and calculation are performed to output to CRT 46 or a printer 46.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chromatography technique such as liquid chromatography and gas chromatography, and more particularly to a chromatogram analysis method (hereinafter referred to as analysis method) and a chromatograph device used therefor, the analysis method and the chromatograph. Data used in the device
Data processing device.

[0002]

2. Description of the Related Art Prior to the description of conventional technology, technical terms will be defined. In the present application, convolution is defined as a convolution integral with respect to two functions x (t) and y (t). Generally, an output waveform from a measuring instrument is an input waveform and a device function (an impulse response in an electric circuit). , In the spectroscope, it becomes a convolution with the slit function). Deconvolution is defined as the inverse operation of convolution. Specifically, it is equivalent to solving an integral equation represented by convolution. The reconvolution means that a device function, that is, a waveform deconvoluted using a function h (t) representing spread is again convoluted using the function h (t).

Conventionally, generally, the output waveform data from the measuring instrument is distorted by the dynamic characteristics of the measuring instrument, that is, the influence of the device function. The same is true for a chromatograph device, in which a device function, that is, a function h (t) representing the spread causes distortion on the chromatogram,
Peak overlap occurs on the chromatogram. In such a case, it is difficult to accurately determine the peak area of each chromatogram.

Conventionally, when the above distortion is received, deconvolution processing is used, and for the overlap of the peaks, a synthetic separation method based on a normal curve fitting method is used, and data processing is performed. It is proposed to decompose and quantify.
As a portable solution method for this, there is a technique described in "Waveform data processing for scientific measurement", edited by Shigeo Minami, published by CQ Publishing Co., Ltd. (1986). Further, many improvement techniques have been proposed, for example, the technique described in JP-A-62-17465 and the technique described in JP-A-63-151851.

The technique described in JP-A-62-17465 is one in which the half-value width on the rising side is approximated by Gaussian, which is a predetermined multiple of the distance from the rising point to the ending point. In the case of overlapping peak waveforms, it is not always possible to replace by such an approximation. Although this technique is effective in reducing the storage capacity, it has a drawback that it is not always effective in accurately determining the peak area when the peak waveforms overlap.

In the technique disclosed in Japanese Patent Laid-Open No. 63-151851, the division of overlapping peaks, which has been conventionally performed by so-called vertical division or skimming division, is divided into two Gaussians and approximated to obtain the minimum two. Although deconvolution processing is performed by a method called multiplication, there is a drawback in that a physically meaningful solution cannot be obtained.

US Pat No. 4807148,
In the technology described in US Pat. No. 4,941,101, etc., a numerical analysis method is used in the deconvolution processing, and the method of Jansson, the Jacobian method,
There is a Gauss-Seidel method and the like, which is advantageous in terms of operation time and the number of memory words, but there is a drawback in expanding discontinuous points of the solution.

[0008]

The various conventional deconvolution processes described above are deconvolution processes when the chromatograms are distorted, and synthetic separation for the case where overlapping peak waveforms of the chromatograms are generated. Provides a solution to each of them, but in the case where an overlapping peak waveform is generated due to distortion, it is considered in detail that the distortion is taken into consideration, and it is considered in detail until the overlapping waveform is separated. Had not been dealt with.

The above-mentioned conventional synthetic separation method for overlapping of chromatogram peak waveforms has the following problems. 1. When assuming a non-linear function such as Gaussian, the validity of selecting that function is questionable. 2. Introducing multi-channel information such as UV spectra requires expensive detectors such as photodiode array detectors. 3. Since the least squares method is used in the peak decomposition, it takes a long calculation time to obtain a solution, and a physically meaningful solution may not be obtained in some cases.

The present invention has been made to solve the above-mentioned problems of the prior art, and improves the separation by correcting the distortion of the chromatogram waveform and sharpening the overlapping peak waveforms. The first object is to provide a chromatogram analysis method in which the peak area is obtained more accurately and the accuracy of quantitative analysis is improved. A second object of the present invention is to provide a chromatograph device using the above chromatogram analysis method. A third object of the present invention is to provide the chromatogram analysis method and a data processing device for the chromatograph device.

[0011]

In order to achieve the above-mentioned first object, the constitution of the present invention relating to a chromatogram analysis method comprises a step of obtaining a detection peak waveform from an analytical sample by a separation column, and the above-mentioned detection. In the chromatogram analysis method including the step of separating the overlapping peaks in the peak waveform and the step of outputting the separated peak waveform, the step of separating the overlapping peaks extends from the detected peak waveform. A method of obtaining the function h (t), and then the function h
(T) is used to deconvolute the detection peak waveform.
And a method of processing the application.

A method of obtaining the spread function h (t) is characterized by setting a reference and using an estimation method. The set reference is characterized in that the overlapping peak waveforms are separated and independent. The spread function h (t) is obtained by controlling at least one of the injected sample, the mobile phase, and the stationary phase to find a peak that is not held, and based on the peak waveform, the spread function h (t). It is characterized by seeking. The spread function h (t) is obtained by preparing at least one of an injection sample, a mobile phase, and a stationary phase, obtaining a peak that is not held, and expanding the spread function h (t) based on the peak waveform. It is characterized by seeking. Spread function h
The method of obtaining (t) is to input parameters that characterize the spread function h (t), and then to expand the spread function h (t).
It is characterized by outputting (t).

The input parameters are the spreading function h
It is characterized by being information that serves as a reference / limitation for determining (t). The information that serves as a reference / limitation is characterized by any one of the analysis initial value, the number of estimated peaks, the size, the width, and the retention time. Spread function h
(T) is a normalized function of the non-retained component in the standard sample. The spread function h (t) is characterized by injecting the non-retained component into the separation column and obtaining it from the detected peak waveform.

In another configuration of the present invention related to the chromatogram analysis method, the step of separating the overlapping peaks in the detected peak waveform and the step of outputting the separated peak waveforms form the overlapping peak waveforms. By the spreading function h (t) used when deconvolution processing is performed so as to be separated / independent, reconvolution processing is performed on each of the separated / independent waveforms, and each waveform after the reconvolution processing is described above. The feature is that it is overlaid with the first overlapping peak waveform.

In order to achieve the above-mentioned second object, the constitution of the present invention relating to a chromatographic apparatus is a means for obtaining a detection peak waveform of a mixture sample by a separation column and an overlap in the detection peak waveform. In a chromatographic apparatus including a means for separating peaks and a means for outputting the separated peak waveform, the means for separating the overlapping peaks is an analysis for obtaining a spread function h (t) from the detected peak waveform. And a processing unit for performing deconvolution processing on the detected peak waveform using the function h (t).

The analysis unit is characterized in that it is configured to set a reference and obtain the spread function h (t) by an estimation method. The standard is characterized in that it is set at a point where the overlapping peak waveforms are separated and independent.
The analysis unit controls at least one of the injected sample, the mobile phase, and the stationary phase, obtains a peak that is not held, and determines the peak.
It is characterized in that the spread function h (t) is obtained based on the waveform. The analysis unit is configured to generate at least one of an injected sample, a mobile phase, and a stationary phase, obtain a peak that is not held, and obtain a spread function h (t) based on the peak waveform. It is characterized by that. The analysis unit uses the spreading function h
It is characterized by comprising an input unit for inputting a parameter characterizing (t) and an output unit for outputting the spreading function h (t).

The input unit is characterized in that it can be inputted with information that serves as a reference / limitation for determining the spread function h (t). The information to be input as reference / limitation is characterized by any one of the analysis initial value, the number of estimated peaks, the size, the width, and the retention time. The spread function h (t) is characterized by being a normalization function of the non-retained component in the standard sample. The spread function h (t) is characterized in that the non-retained component is injected into the separation column and is obtained from the detected peak waveform.

Another configuration of the present invention relating to a chromatographic apparatus is that the means for separating overlapping peaks in the detection peak waveform and the means for outputting the separated peak waveforms separate the overlapping peak waveforms. .Spread function h used when performing deconvolution processing so as to be independent
According to (t), each of the separated and independent waveforms is subjected to reconvolution processing, and each waveform after the reconvolution processing is overlaid on the first overlapping peak waveform.

In order to achieve the above-mentioned third object, the constitution of the present invention relating to the chromatogram analysis method and the data processing device for a chromatograph apparatus is such that an A / D conversion section, a memory section, a control section, In a data processing apparatus used in a chromatogram analysis method and a chromatograph apparatus, which includes an interface section and an output device, a function h (t) representing a spread from a peak waveform detected by the chromatogram.
Of the detected peak waveform and the function h
(T) is used to deconvolute the detection peak waveform.
The chromatogram after the deconvolution process is output.

More specifically, the peak of the chromatogram is widened due to various causes. For example, the sample zone is expanded by flowing the sample through a flow path such as a pipe or a flow cell of a detector. This is called the spread outside the column. The contribution of the spread, typified by the spread outside the column, is convolved with the original peak waveform.
Is calculated and appears in the detected waveform. That is, if the spread function is known, the original peak waveform can be calculated by performing deconvolution operation on the detected waveform.

There are various possible methods for determining the spread function h (t). The methods are roughly classified into two methods: an analytical mathematical h (t) having several parameters that characterize the method and the method obtained experimentally from the detected data.
There is a method to assume. The former can be considered as a method in which h (t) is known, and the latter can be considered as a method in which h (t) is unknown.

As a method for obtaining the above-mentioned h (t) from the measurement, for example, in order to obtain the spread outside the column, the column is replaced with a joint, a sample is injected, and the waveform measured by the detector is referred to as h (t). There is a way to consider. To add a spread in the column that does not depend on the type of components in the sample, inject a sample that is not held and change its peak waveform to h
It can be regarded as (t).

Other methods for obtaining peaks that do not retain are to use a solvent having a very strong elution capacity for the eluent, to prepare a column packing material that does not modify the functional group, and to not hold it. Conceivable. It is also effective to add non-retained components to the standard sample and obtain non-retained peaks from the chromatogram of the standard sample.

When h (t) is unknown, the spreading function h corresponding to a part of the peak width is premised on the following points.
(T) can be deconvoluted, and can be accurately quantified based on the peak area from a chromatogram having a sharper peak.

The precondition is that the peak waveform to be detected is originally a delta function or a very sharp rectangular concentration distribution function, and a spread function is subjected to a convolution operation. It is believed that there is. Here, the spread function is almost Gaussian, and has a waveform that is slightly modified in a telling or reading manner. In other words, the spreading function itself is a convolution with a correction function of tailing or reading for Gaussian.
It is considered that the operation is being performed.

Gaussian has the additivity that when two Gaussians are subjected to a convolution operation, it is also Gaussian. As described above, by performing deconvolution operation on the Gaussian forming a part of the spread function, it is possible to calculate the peak that is closer to the sharper original waveform.

[0027]

The function of each of the above technical means is as follows.
According to the configuration of the first invention, a known h (t) or Gaussian is rationally selected from the detection chromatogram and the deconvolution operation is performed, so that a sharper peak than the overlapping peak of the detection chromatogram can be obtained. When the chromatogram having peaks is separated / independent and each of the separated / independent peaks is integrated, the peak area is accurately obtained, and accurate quantification can be performed. In the deconvolution operation, unlike a method of least squares of a non-linear function, for example, a physically meaningful solution can always be obtained with a short calculation time.

In the deconvolution operation, when the overlapping peaks are separated / independent, the chromatogram is observed and the validity of the operation is judged, but skill is required. Therefore, the spread function h (t) used in the deconvolution calculation is used to perform the reconvolution calculation on each independent peak, and each peak forming the first overlapping peak can be reproduced. The validity of the deconvolution calculation can be accurately determined by superimposing each peak after the reconvolution calculation and the first overlapping peak.

According to the structure of the second invention, the chromatogram analysis method of the first invention can be implemented with a simple structure by omitting an expensive detector such as a photodiode array detector. According to the configuration of the third invention, the data of the chromatogram analysis method of the first invention and the data of the chromatogram analysis device of the second invention can be processed accurately and economically.

[0030]

【Example】

[Embodiment 1] An embodiment of the present invention will be described below with reference to FIGS. 1 to 11. FIG. 1 is a block diagram showing a configuration of a microbore HPLC system used in a chromatogram analysis method (hereinafter referred to as an analysis method) according to an embodiment of the present invention, and FIG. 2 shows a chromatogram in the analysis method of FIG. FIG. 3 is a schematic explanatory view of the spread mechanism of the chromatogram in the analysis method of FIG. 1, and FIG. 4 is a deconvolution in the analysis method of FIG.
FIG. 5 is a flow chart of processing steps according to the analysis method of FIG. 1, and FIG. 6 is a plot plot of chromatograms in the analysis method of FIG.

FIG. 7 is a diagram showing a chromatogram of amino acid analysis by the analysis method of FIG. 1, FIG. 8 is a diagram showing a chromatogram of glycohemoglobin analysis by the analysis method of FIG. 1, and FIG. A processing flow chart when the spread function is unknown in the analysis method of FIG. 1, FIG. 10 is a diagram showing a chromatogram of the catecholamine analysis in the analysis method of FIG. 1, and FIG. 11 is the analysis of FIG. FIG. 8 is an explanatory diagram of overlapping drawing by reconvolution calculation in the method.

A microbore HPLC system is used to carry out the chromatogram analysis method according to the first invention. FIG. 1 shows the overall configuration of a microbore HPLC system. In FIG. 1, 40 is a pump, 41 is a column, 42 is a detector, 43 is a sampler, 44 is a control unit, 45 is a data analysis unit, 46 is a CRT display unit, 47 is a printer, 49 is an eluent, 51 Is an unknown sample, 52 is a standard sample, and 54 is an input unit.

The respective constituent parts of the present system use the usual chromatogram analysis method except for the data analysis part 45 (which will be described later in [Example 2]). Therefore, the detailed description is complicated and will be omitted, and will be briefly described.

In the microbore HPLC system,
The spread outside the column greatly affects the separation and cannot be ignored. The pump 40 sends the eluent 49 according to a command from the controller 44. The sampler 43 having a movable needle valve injects the unknown sample 51 or the standard sample 52 into the flow path to the column 41. The sample is sent to the column 41 together with the eluent 49, the contained components are separated and developed, and detected by the detector 42.

The detected chromatogram is stored in the data analysis unit 45. FIG. 2 is the detected chromatogram. Since the non-retained component is added to the standard sample 52, it is detected as the non-retained peak 61. The peak 62 to be eluted next is one component of the standard sample, is retained in the column unlike the peak 61, and the peak width is slightly wide.

The mechanism by which the peak waveform is formed will be described with reference to FIG. FIG. 3 is an explanatory diagram of a mechanism of spreading the chromatogram in the analysis method according to the embodiment of FIG. In FIG. 3, the same reference numerals as those in FIG. 1 are the same parts in FIG. The upper (a) diagram is a block diagram showing the sample channel, and the lower (b) diagram shows the concentration of the sample in the sample channel of the upper (a) diagram.

The sample containing a single component is injected into the channel by the sampler 43. Sample concentration C in the flow path at this time
The spatial distribution diagram of is a rectangular distribution diagram 71 where the flow direction is x. This rectangular distribution diagram 71 expands when it passes through the column 41. The stage after passing through the column 41 has a Gaussian shape as shown in the distribution diagram 72.

Further, when the detector 42 detects the Gaussian concentration distribution, the concentration distribution is expanded by the flow passage piping and the flow cell of the detector. It has a wide distribution shape.

If this is described by a mathematical expression, it can be expressed by a model like the expression (1).

[Equation 1] In the above equation, the detected waveform at time t is y (t), the original rectangular waveform at the time of sample injection is r (t), and the spread function is h (t). The above formula is the detected waveform y
(T) indicates that it is a convolution of the original rectangular waveform r (t) and the spreading function h (t).

The spread of the shape of the peak may be described above, but considering the retention time, it is considered that another Dirac delta function is subjected to the convolution operation. This is shown in equation (2).

[Equation 2]

In the above equation, EMG (t) is Expo
mentally Modified Gaussi
n, σ, τ, and t _R are parameters of EMG (t), δ (t) is a delta function, and m (t) is a correction function.
For the spreading mechanism, see “DYNAMICS
OF CHROMATO GRAPY ", JC Gid
by Dings, published by Marcel Dekker, New
It is described in detail in York, 1965, and will be referred to here.

The spread function h (t) is formed by the influence from both the spread inside the column and the spread outside the column. In other words, it is a convolution of the spreading function inside the column and the spreading function outside the column.

Paying attention to the non-holding peak 61 in FIG.
Explaining with reference to FIG. 3, the rectangular waveform 71 at the time of injection passes through the column 41 and is expanded to the Gaussian waveform 72, and is affected by the expansion outside the column to become the waveform 73, which becomes the peak 61. It has been detected.

On the other hand, the holding peak 62 is expanded by a similar mechanism to form a peak, but since it is held in the column 41, it is a slightly wider peak.
The width is wide. That is, the spreading function that affects the peak 62 is a convolution of the spreading function common to the peak 61 and the surplus spreading function other than that.

In this case, the influence of the spreading function of the peak 61 can be removed from the spreading function of the peak 62, and only the surplus spreading function can be obtained. This processing is called deconvolution processing.

The deconvolution operation is shown in FIG.
It is effective for overlapping peaks such as the peak 64 and the peak 65 shown in FIG. This will be described with reference to FIG. Figure 4
Describes a deconvolution operation for the overlapping peaks shown in FIG.

When the spread function of the peak 61 waveform is deconvoluted with respect to the peak 64 waveform and the peak 65 waveform, the separated excess peaks as shown in FIG. 4 are obtained. Is obtained, and more accurately each peak
The area of ku can be calculated. In practice, a normalization function that normalizes the peak waveform 61 to area 1 is used for deconvolution calculation. Normalization is done to preserve the peak area.

Strictly speaking, this normalization function is
A standardized convolution of the rectangular function r (t) and the spreading function h (t) affecting the peak waveform 61 when 1 is injected.
It is an option. In practice, the peaks other than the peak waveform 61 are also affected by the rectangular function, so that there is no problem even if the deconvolution operation is performed using the standardization function of the peak 61 waveform.

The above deconvolution procedure of FIG. 4 will be described with reference to the block diagram of the microbore HPLC system of FIG. 1 and the flowchart of the detection waveform processing shown in FIG. First, standard sample 5 with non-retained components added
2 is injected, and waveform processing is started in step 31.

Next, in step 32, the non-holding peak 61 is identified. This is automatically identified as a peak of a certain size or more that appears at the first time of the chromatogram, or the operator specifies the time from the input section 54, or CR
It can be designated by using the mouse from the chromatogram displayed at T46 and picking up the peak 61.

In step 33, the area of the non-holding peak 61 is normalized to 1. At this time, the standardization function can be displayed on the CRT 46. In step 34, the operation
Set the start point and end point of the time when there is an overlap peak that you want to accurately quantify. On the CRT 46, as shown in the left diagram of FIG. 4, it is possible to display a chromatogram in which only this portion is enlarged. Here, the operator can also set all times of the chromatogram.

In step 35, the deconvolution process is performed within the set time interval using the normalizing function. In step 36, the chromatograms before and after the treatment are C
It is displayed on the RT 46 as shown on the right side and the left side of FIG. Further, as shown in FIG. 6, it is also possible to plot a chromatogram overlaid on the printer 47.

In step 37, the figure shown in FIG.
The integration is executed for the peak on the right side of, and quantitative calculation is performed. If the peaks in the chromatogram after this treatment still overlap, the area is calculated by vertical division as usual. Here, it is also possible to perform area division by the least square method with a non-linear function such as Gaussian or EMG. CRT4 here
6 or outputs the quantitative value to the printer 47. The process ends at step 38.

Although the spreading function for deconvolution processing is substituted by the normalization function of the peak waveform of the non-holding component in the standard sample 52, the spreading function can be obtained by other than this method. Instead of adding to the standard sample, only the non-retained component can be injected to obtain the spread function in advance, and the peak waveform can be obtained.

In the case of reverse phase chromatography, an eluent of 100% organic solvent such as methanol, acetonitrile, THF, chloroform is flowed to inject weakly retained components,
A non-holding peak can be obtained. In ion exchange chromatography, for example, in the case of an amino acid analyzer, a regenerating solution having a high pH is caused to flow and phosphoserine or the like, which has a weak retention, is injected to obtain its peak waveform.

Further, by operating the column packing material, the non-retained
You can also get Ion exchange chromatography
Then, a non-retaining peak can be obtained by preparing a filler containing only a carrier having no modified ion exchange group.

An amino acid analyzer will be mentioned as an example of the post-column reaction LC. This system cannot ignore the spread outside the column. The spread outside the column can be estimated by replacing the column with a joint and observing the waveform of the non-holding peak as a spread function.

In the chromatogram detected as shown in FIG. 7, threonine (Thr) and serine (Ser) were detected.
The overlapped portion was 14%, while the deconvolution-treated chromatogram improved to 4%, which is 1/3 or less. This processed chromatogram corresponds to the chromatogram when post-column reaction LC was detected immediately after the column.

As an example of removing the common part of the spreading function,
3. Glycohemoglobin analyzer for 3 minutes.
The spread function can be estimated from the peak waveform of HbF shown on the left side of FIG. This is because the peak width of HbA1c is
This is because it is considered to be slightly wider than the width of HbF in consideration of the order of retention times.

The waveform of HbF, which is an isolated peak, is standardized from the detection chromatogram on the left side of FIG. 8 to obtain the spread function. By using the normalization function to perform deconvolution processing on the overlapping peaks of unstable and stable HbA1c, a chromatogram with improved separation as shown on the right side of FIG. 8 can be obtained.

In the deconvolution process, it is not necessary to directly display the processed chromatogram in this case, but in order to improve the quantification accuracy, it is a very effective technique even in a general HPLC glycohemoglobin analyzer. is there.

Next, the processing to be used before the deconvolution operation to be performed correctly will be described. Noise 67 and drift 68 shown in FIG. 2 interfere with the deconvolution operation. If these are subjected to deconvolution processing as they are, problems occur such that they are amplified as pseudo peaks, and the peaks to be deconvolution processed are not processed correctly.

First, as the noise removal processing, generally, a method of adding some data points of the detected chromatogram and reducing it is adopted. In addition to this, a smoothing method or a known method of removing high frequency components can be used.

As the drift removal process, generally, a method of connecting valleys between peaks with a straight line or forcibly drawing a straight line and subtracting the base line from the detected chromatogram is adopted. In addition, known methods for removing low-frequency components, methods for removing low-order polynomial components using calculus, methods for searching base lines from differential feature points, methods for using neural networks, etc. There is.

Up to this point, the spreading function has been obtained from the measurement data, but even if the spreading function is unknown, Gaussian can be deconvolved. As described above, the peak waveform of the detection chromatogram is (1)
As shown by the equation, it can be expressed by the convolution of the spreading function h (t) and the rectangular waveform r (t).

This spread function h (t) is obtained by the above-mentioned "DY
NAMICS OF CHROMATO GRAPY "
J. C. According to Giddings, no matter what kind of physical or chemical interaction the spread in the column is, the spread outside the column has a Gaussian-like shape even if it is large enough to be ignored.

Even if h (t) is slightly distorted from Gaussian in the manner of reading or tailing, Gaussian G (t) and the correction function m are obtained as shown in the following equation (3).
It can be described as a convolution with (t).

[Equation 3]

In the above equation, Gaussian is G (t), the correction function is m (t), and σ is the standard deviation. As an example, h
If (t) is a tailing peak that can be represented by EMG (t), a correction function can be obtained analytically and mathematically as in the above equation (2).

Further, the overlapping peak can be expressed by the equation (4).

[Equation 4] Here, σ ₁ and σ ₂ are standard deviations, respectively.

Since Gaussian has additivity as described above, it can be decomposed into two Gaussian convolutions, and therefore has a relation as shown in the following expression (5).

[Equation 5]

That is, the two peak waveforms represented by the equation (4) can be subjected to deconvolution calculation using Gaussian. At this time, the Gaussian has a standard deviation σ ₀ smaller than the standard deviations σ ₁ and σ ₂ of the two peaks.
Must have a narrow waveform with.

In this way, it is possible to obtain a peak whose width is narrower than the detected overlapping peak. In the end, y
When (t) is deconvoluted by Gaussian G (t, σ ₀ ), the chromatogram can be represented by the convolution with the correction function as it is with a narrower Gaussian.

[0073]

[Equation 6] As shown in [] of the above equation, a peak with a narrower peak width than σ ₁ and σ ₂ is obtained. More specifically, the rectangular waveform at the time of injection is subjected to convolution operation by this correction function.

The process when the spread function h (t) is unknown will be described with reference to FIG. The process starts at step 71. Step 72 removes noise drift from the detected chromatogram and prepares for deconvolution processing. In step 73, it is input whether the deconvolution process is to be performed manually or automatically.

In the automatic case, in step 81, the peak width of the detected chromatogram is viewed as a whole, and a Gaussian having a standard deviation slightly smaller than the minimum width is set as a spreading function. It is standardized to area 1. Here, when the peak width is twice or more different between the peak having a short retention time and the peak having a long retention time, the deconvolution operation can be performed by dividing the time.

Here, it is possible to use the Fourier transform to automatically find the peak width. In step 76, deconvolution processing is executed. In step 77, it is determined whether the processed chromatogram has not diverged or is less than the negative lower limit value. Further, it is determined whether or not the non-separated peak forms a valley between the peaks.

If the judgment here is acceptable, the process proceeds to step 79. If the determination is not successful, the process returns to step 81 again, the standard deviation is adjusted with reference to the determination, and deconvolution processing is performed. If the determination is successful, the process chromatogram is displayed and the quantitative result is displayed in step 79. Process 80
Ends with.

In the case of manual operation, an input operation is performed in step 74. Generally, the standard deviation of Gaussian to be deconvoluted is entered. In addition, the start and end points of the processing section can be entered. It is also possible to search for an optimal Gaussian standard deviation in order to improve the separation of specific overlapping peaks.

In this case, the initial value of the standard deviation, the number of estimated peaks, the size, the width, the holding time, etc. can be input as reference values. As a limit value, the number of peaks is up to 3, or the width of the overlapping peaks is 0.2 minutes or more.
You can also enter 4 minutes or less.

In step 75, the information input in step 74 is calculated to obtain the set value for deconvolution processing,
A reference value for judgment is calculated. Deconvolution at step 76
Solution processing. A determination is made in step 77. If it fails, the reason is displayed in step 78, and re-input is prompted in step 74. If it passes, the processed chromatogram and the quantitative result are displayed in step 79. The process ends at step 80.

The raw 10-minute chromatogram obtained by the catecholamine analyzer is shown in the upper part (a) of FIG. This is an example of analysis of biological samples such as plasma and urine. Numerous peaks often appear when analyzing biological samples. When this is subjected to deconvolution with an appropriate Gaussian, a chromatogram as shown in the middle row (b) is obtained. Each peak is completely separated up to the base line.

However, even if this is displayed as it is, it is difficult to understand the meaning of the processed chromatogram. Rather, this may be a known display method shown in (c) at the bottom. Further, it is considered that only the quantitative value is output and the operator is not made aware of the deconvolution process.

Although one embodiment of the first invention has been described above, the present invention is not limited to the above embodiment, and various modifications can be considered. Another method according to the present invention will be described. FIG. 11 is a diagram for explaining overwriting by deconvolution and reconvolution in the analysis method according to the first invention.

FIG. 11A shows the detected original waveform. Deconvolution to this original waveform
Processing is performed to separate and separate each waveform peak. This state is shown in FIG. 11 (2). The separated / independent waveform peaks are reconvoluted by the same spreading function h (t) as when deconvoluting. Reconvolution The calculated waveform is overlaid on the detected original waveform. FIG. 11 (3) shows this state.

[Embodiment 2] An embodiment of the chromatographic analysis apparatus according to the present invention will be described. FIG. 12 is a block diagram showing the configuration of a chromatographic analysis device which is another embodiment of the present invention. Regarding the overall configuration of the chromatographic analysis device according to the second invention, the microbore HPLC system of FIG. 1 has been described in [Example 1], which is troublesome and will not be described again. Only explain.

The data analysis unit 45 comprises a noise / drift removal mechanism, a spread function analysis mechanism, a deconvolution operation mechanism, a quantitative calculation mechanism, and a determination mechanism. Although not shown in the figure, it goes without saying that an A / D converter, a storage device, an interface unit and the like are also provided. Among the above-mentioned configurations, the noise / drift removing mechanism, the quantitative calculation mechanism, and the determining mechanism are known techniques, but will be described together in order. The respective mechanical units are connected by a bus.

The noise / drift removing mechanism is provided with a smoothing mechanism, a base line calculating mechanism signal, a fast Fourier transform mechanism, a neural network mechanism, a differentiation / integration mechanism, and a fuzzy inference mechanism. The smoothing mechanism uses the moving average method, the Savitzky-Golay method, and the measurement data group of the chromatogram from the detector 43.
Fitting to a smooth curve by either the Kawata-Minami method or the frequency domain method, the base line calculation mechanism determines the base line by an appropriate algorithm.

The fast Fourier transform mechanism removes noise, which is a high frequency component, and base line fluctuation, which is a low frequency component, based on the frequency distribution. Further, the neural network mechanism learns the drift shape in advance and removes the drift from the measured data according to a predetermined coupling constant.

The differentiation / integration mechanism removes the base line component represented by a low-order polynomial by differentiation / integration. A fuzzy inference mechanism smooths the arranged measured data point group according to a predetermined coupling constant. In this way, chromatogram noise and drift from detector 43 is eliminated.

The spread function analysis mechanism is based on Gaussi.
An an-generation mechanism, an area 1 normalization mechanism, and a detection peak waveform setting mechanism are provided. In the case of an analytical mathematical spread function, Gaussian generation mechanism generates Gaussian. In the case of the measured spread function, the peak waveform measured by the area 1 normalization mechanism is standardized, and the standardized measured waveform by the detected peak waveform setting mechanism is used as the spread function. In this way, a measured or analytical mathematical spreading function is generated.

The deconvolution operation mechanism includes a time interval setting mechanism, a spread function setting mechanism, and a deconvolution processing mechanism, and the time interval setting mechanism sets the interval for deconvolution processing, The spread function setting mechanism sets a spread function from the generated actual measurement or analytical mathematical spread function. The deconvolution processing mechanism performs deconvolution processing on the set overlapping waveform in the deconvolution processing section using the spreading function, and separates and separates the overlapping waveform.

The quantitative calculation mechanism includes an integration mechanism, and the integration mechanism calculates the areas of the separated and independent peak waveforms. Thus, the quantification is calculated based on the peak area or height. The quantitative result is CRT
46, and is output to the printer unit 47.

The determination mechanism for making various determinations searches for the discriminant calculation mechanism for calculating the value of the discriminant used for determining the success of the deconvolution process, and the Gaussin used for the deconvolution process. A limit value calculation mechanism for calculating a value that is sometimes a limit and a spread function search mechanism for searching for the optimum Gaussin according to a predetermined coupling coefficient are provided, and each mechanism can perform a predetermined function at each processing stage. I support you.

The functions of the control unit 44, the data analysis unit 45, the CRT unit 46, the printer unit 47, etc. in FIG. 12 are integrated to form an A / D conversion unit, a memory unit, a control unit, and an interface unit. , And an output device, which calculates and outputs a function h (t) representing the spread from the detected peak waveform of the coatgram, and uses the detected peak waveform and the function h (t) to detect the detected peak waveform. It is possible to configure a data processing device used for a chromatogram analysis method and a chromatograph device, characterized in that the deconvolution process is performed and the chromatogram after the deconvolution process is output.

[0095]

As described above in detail, according to the structure of the present invention, first, the distortion of the chromatogram waveform is corrected, and the overlapping peak waveforms are sharpened to be separated. It is possible to provide a chromatogram analysis method since the peak area is improved, the peak area is obtained more accurately, and the accuracy of quantitative analysis is improved. Secondly, it is possible to provide a chromatograph device used for carrying out the chromatogram analysis method. Thirdly, it is possible to provide a data processing device used in the chromatogram analysis method and the chromatograph device.

[Brief description of drawings]

FIG. 1 is a block diagram showing the configuration of a microbore HPLC system used in an analysis method according to an embodiment of the present invention.

FIG. 2 is a diagram showing a chromatogram in the analysis method of FIG.

FIG. 3 is a schematic explanatory view of a spread mechanism of a chromatogram in the analysis method of FIG.

FIG. 4 is an explanatory diagram of deconvolution calculation for overlapping peaks of the chromatogram in the analysis method of FIG.

5 is a flowchart of processing steps according to the analysis method of FIG.
It is

FIG. 6 is an overlay plot diagram of chromatograms in the analysis method of FIG.

FIG. 7 is a diagram showing a chromatogram of amino acid analysis by the analysis method of FIG.

FIG. 8 is a diagram showing a chromatogram of glycated hemoglobin analysis by the analysis method of FIG.

FIG. 9 is a processing flow chart when the spread function is unknown in the analysis method of FIG.

FIG. 10 is a diagram showing a chromatogram of catecholamine analysis in the analysis method of FIG.

FIG. 11 is an explanatory diagram of overwriting by a re-convolution operation in the analysis method of FIG.

FIG. 12 is a block diagram showing the configuration of a chromatographic analysis device according to another embodiment of the present invention.

[Explanation of symbols]

 40 ... Pump 41 ... Column 42 ... Detector 43 ... Sampler 44 ... Control unit 45 ... Data analysis unit 46 ... CRT display unit 47 ... Printer 49 ... Eluent 51 ... Unknown sample 52 ... Standard sample 54 ... Input unit

Claims (23)

[Claims] 1. A detection peak from an analytical sample by a separation column.
In a chromatogram analysis method that includes a step of obtaining a waveform, a step of separating overlapping peaks in the detected peak waveform, and a step of outputting the separated peak waveform, the step of separating the overlapping peaks is , A method for obtaining a spread function h (t) from the detected peak waveform, and a method for deconvolution processing the detected peak waveform using the function h (t). Chromatogram analysis method. 2. The chromatogram analysis method according to claim 1, wherein a method for obtaining the spread function h (t) is based on an estimation method by setting a standard. 3. The chromatogram analysis method according to claim 2, wherein the reference is a point at which overlapping peak waveforms are separated and independent. 4. A method of obtaining a spreading function h (t) is performed by obtaining a peak that controls and holds at least one of an injected sample, a mobile phase, and a stationary phase, and based on the peak waveform, the spreading function. The method according to claim 1, wherein h (t) is obtained.
The described chromatogram analysis method. 5. A method for obtaining the spread function h (t) is performed by obtaining a peak that does not hold at least one of an injected sample, a mobile phase and a stationary phase, and based on the peak waveform, the spread function. The method according to claim 1, wherein h (t) is obtained.
The described chromatogram analysis method. 6. The method for obtaining the spreading function h (t), comprising inputting a parameter characterizing the spreading function h (t), and then outputting the spreading function h (t). Chromatogram analysis method described in 1. 7. The input parameter is a spreading function h
The chromatogram analysis method according to claim 6, which is information serving as a reference and a limit for determining (t). 8. The information used as reference / limitation is the analysis initial value,
The chromatogram analysis method according to claim 7, wherein the estimated peak number is any of the number, size, width, and retention time of the peaks. 9. The spread function h (t) is a normalization function of non-retained components in a standard sample.
9. The chromatogram analysis method according to any one of 8 to 8. 10. The chromatogram analysis according to claim 4, wherein the spread function h (t) is obtained from a detection peak waveform of a non-retained component injected into a separation column. Method. 11. A step of obtaining a detection peak waveform from a sample to be analyzed by a separation column, a step of separating overlapping peaks in the detection peak waveform, and a step of outputting the separated peak waveform. In the chromatogram analysis method, the step of separating the overlapping peaks in the detection peak waveform and the step of outputting the separated peak waveforms,
Spreading function h used when deconvolution processing is performed to separate and separate overlapping peak waveforms.
The chromatogram analysis method according to (t), wherein each of the separated and independent waveforms is subjected to a reconvolution process, and each waveform after the reconvolution process is overlaid on the first overlapping peak waveform. 12. A means for obtaining a detection peak waveform from a mixture sample by a separation column, a means for separating overlapping peaks in the detection peak waveform, and a means for outputting the separated peak waveform. In the chromatographic apparatus, the means for separating the overlapping peaks includes an analysis unit that obtains a spread function h (t) from the detected peak waveform, and a deconvoluted detected peak waveform using the function h (t). A chromatographic apparatus, comprising: 13. The chromatograph apparatus according to claim 12, wherein the analysis unit is configured to set a reference and obtain the spread function h (t) by an estimation method. 14. The chromatograph device according to claim 13, wherein the reference is set to a point where the overlapping peak waveforms are separated and independent. 15. The analysis unit obtains a peak that controls and holds at least one of the injected sample, mobile phase, and stationary phase, and obtains a spreading function h (t) based on the peak waveform. 13. The chromatograph device according to claim 12, wherein the chromatograph device is configured as described above. 16. The analyzing unit obtains a peak that does not hold at least one of an injected sample, a mobile phase and a stationary phase, and obtains a spread function h (t) based on the peak waveform. 13. The chromatograph device according to claim 12, wherein the chromatograph device is configured as described above. 17. The analysis unit comprises an input unit for inputting parameters characterizing the spread function h (t) and an output unit for outputting the spread function h (t). 12. The chromatograph device according to item 12. 18. The chromatograph apparatus according to claim 17, wherein the input unit is configured to be able to input information that serves as a reference and a limit for determining the spread function h (t). 19. The chromatograph apparatus according to claim 18, wherein the input information is any of an analysis initial value, the number of estimated peaks, a size, a width, and a retention time. 20. The chromatographic apparatus according to claim 12, wherein the spread function h (t) is a normalization function of non-retained components in a standard sample. 21. The spread function h (t) is configured so as to be obtained from a detection peak waveform by injecting a non-retained component into a separation column.
6. A chromatograph device according to any one of 6. 22. A means for obtaining a detection peak waveform from a mixture sample by a separation column, a means for separating overlapping peaks in the detection peak waveform, and a means for outputting the separated peak waveform. In the chromatographic device, the means for separating overlapping peaks in the detection peak waveform and the means for outputting the separated peak waveform are
Spreading function h used when deconvolution processing is performed to separate and separate overlapping peak waveforms.
According to (t), each of the separated and independent waveforms is subjected to reconvolution processing, and each waveform after the reconvolution processing can be superimposed and drawn on the first overlapping peak waveform. Graph device. 23. A coattogram analysis method comprising an A / D conversion section, a memory section, a control section, an interface section, and an output device, and a data processing apparatus used in a chromatograph apparatus, wherein the coattogram is provided. Of the detected peak waveform, the function h (t) representing the spread is calculated and output, and the detected peak waveform and the function h (t) are used to deconvolution the detected peak waveform. -A data processing device used in a chromatogram analysis method and a chromatograph device, which outputs a chromatogram after the treatment.