JP2018013427A - Method for correcting base line including step - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a processing method capable of obtaining a stable quantitative result even if there is a step in a chromatogram.SOLUTION: A method for smoothing a chromatogram obtains a differential curve of the chromatogram, excludes a differential signal change including only one of a maximum value and a minimum value by spike noise processing, integrates the obtained differential curve in a minute time section, and thereby re-converts the original into a chromatogram.SELECTED DRAWING: Figure 7

Description

  The present invention relates to a processing method for processing a step generated on a baseline of a chromatogram and obtaining a stable quantitative result.

  Chromatography is used in a wide range of fields for quantitative and qualitative purposes. In order to accurately perform quantification and qualification, it is important to acquire a stable chromatogram, and for this purpose, it is necessary to stabilize the baseline.

  The baseline in liquid chromatography is superimposed with noise from various factors such as pulsation of the pump that pumps the liquid, optical noise of the detector, electrical noise, noise from the eluent, and noise that depends on the environment. .

  When short wave noise derived from a liquid feeding period such as a liquid feeding pump as shown in FIG. 1a is superimposed, the “simple moving average method” which is a general mathematical method is effective. As shown in FIG. 1b, smoothing can be easily performed by performing smoothing processing of 101 points by the simple moving average method.

  On the other hand, when short-period noise as shown in FIG. 2a is superimposed and a step is generated on the baseline, the step is smoothed as shown in FIG. 2b even if smoothing processing is performed by the simple moving average method. I can't do it.

  Such a step-by-step variation in the baseline may be caused by sudden bubbles in the detector cell, slight bubbles in the pump while the eluent is being sent, or some vibration on the device. It is not uncommon that it is caused by uncertain factors such as unintentional electrical noise, power supply voltage fluctuations, or the like.

  The present invention provides a technique for smoothing a step and noise while maintaining quantitativeness even in a chromatogram having a step.

  As a result of intensive studies, the present inventors have found a processing method capable of obtaining a stable quantitative result by smoothing a step generated on the baseline of a chromatogram, and complete the present invention. It came to.

  Hereinafter, the present invention will be described in detail.

  The present invention acquires a differential curve of a chromatogram, eliminates a differential signal change having only one of a maximum value and a minimum value by spike noise processing, and integrates the obtained differential curve in a minute time interval. The method for smoothing a chromatogram is characterized by re-converting into a chromatogram.

  Further, the above-described method for smoothing a chromatogram may be applied to a differential curve in the entire time domain of the chromatogram, or may be applied only to a differential curve in the time domain where a step of the chromatogram is generated.

  Further, the present invention can be used without limiting the chromatography method, but can be particularly preferably used for a chromatogram obtained from liquid chromatography.

  According to the present invention, it is possible to provide a processing method capable of obtaining a stable quantitative result even in a chromatogram having a step.

  Hereinafter, embodiments of the present invention will be described in detail.

  As a method for removing spike noise of the present invention, various mathematical methods such as a method using Fourier transform, a Savitzky-Golay method, a 50th percentile filter method, and a median filter method can be mentioned. If there is no particular limitation. Furthermore, when the fluctuation (spike noise) derived from the step on the differential curve is clear, the spike noise may be eliminated by a method.

  In order to explain the embodiment of the present invention in an easy-to-understand manner, each step will be described based on a pseudo-chromatogram combined with a normal distribution function.

  FIG. 3a is a pseudochromatogram with two normally distributed peaks. The horizontal axis corresponds to the elapsed time, and the vertical axis corresponds to the absorbance (mABU). FIG. 3b is a simulated chromatogram with steps of 0.5 mABU from 0.5 to 0.7 minutes and an output of 1.0 mABU from 3.0 to 3.5 minutes. All were produced at a sampling interval corresponding to 50 msec in elution time.

(Step 1)
First, the amount of change, that is, the derivative of the output per hour of the chromatogram is acquired (see FIG. 4). As a result, the normal peak has a certain width and takes the form of a normal distribution, so in the peak part, the differential value gradually increases from zero, passes through the maximum value, decreases, zero, decreases, After passing through the local minimum, it increases and approaches zero. The time when the differential value between the maximum value and the minimum value becomes zero is almost the peak apex (elution time).

  On the other hand, in the portion where the step is generated, the amount of change in output per time becomes extremely large. In the portion where the output rapidly increases, the differential value increases rapidly from zero, passes through the maximum value, decreases rapidly, and becomes zero. In the portion where the output decreases rapidly, the differential value decreases rapidly from zero, and the minimum value increases rapidly and becomes zero. That is, the differential curve of the step portion has a spike noise shape, and thus has only one of the maximum value and the minimum value.

(Step 2)
The differential curve obtained in step 1 is smoothed by a mathematical method such as a method using a Fourier transform, a Savitzky-Golay method, a 50th percentile filter method, a median filter method, etc. Acquire the removed differential curve.

(Step 3)
For the differential curve obtained in step 2, the area is calculated in a minute time interval, and the total area of the minute intervals is calculated, that is, integrated. This reconstructs the output over time, ie the chromatogram. The reconstructed chromatogram obtained by the series of operations is in a state in which the step is eliminated.

  The present invention can effectively smooth not only the step on the baseline but also the step generated while the peak component is being eluted.

  FIG. 5 is a pseudo-chromatogram in which a peak of 2 mABU is added to 4.9 minutes while the peak is eluted from 4.7 to 5.3 minutes. All were produced at a sampling interval corresponding to 50 msec in elution time.

(Step 1)
First, the amount of change in the output per hour of the chromatogram, that is, the derivative is obtained. As a result, the normal peak has a certain width and takes the form of a normal distribution, so in the peak part, the differential value gradually increases from zero, passes through the maximum value, decreases, zero, decreases, After passing through the local minimum, it increases and approaches zero. The time when the differential value between the local maximum and the local minimum becomes zero is almost the peak apex (elution time). In the meantime, in the portion where the output increased rapidly (4.9 minutes), the differential value increased rapidly, passed through the maximum value, decreased rapidly, and the region where the peak was eluted and the peak It can be seen that there is a large difference in the differential curve even in the stepped portion in the eluted region (see FIG. 6).

(Step 2)
The differential curve obtained in step 1 is smoothed by a mathematical method such as a method using Fourier transform, a Savitzky-Golay method, a 50th percentile filter method, a median filter method, etc. Acquire the removed differential curve.

(Step 3)
For the differential curve obtained in step 2, the area for the differential value is calculated in a minute time interval, and the total area of the obtained minute intervals is calculated, that is, integrated. This reconstructs the output over time, ie the chromatogram.

  As described above, the method of the present invention can perform smoothing not only on the step in the region where the peak component is not eluted, but also on the step in the region where the peak component is eluted. I understand that there is.

It is the figure which showed the example which smoothed the signal containing short waveform noise by the simple moving average method. It is the figure which showed the example which smoothed the signal containing short waveform noise and a baseline level | step difference by the simple moving average method. a. It is the figure which showed the simulation chromatogram without a base fluctuation | variation by a normal function. b. It is the figure which showed the simulation chromatogram with which the level | step difference has arisen by the normal function. It is the figure which showed the procedure which smoothes the chromatogram of FIG. 3b by the method of this invention. It is the figure which showed the simulation chromatogram which the level | step difference has arisen in the peak elution area | region by a normal function. It is the figure which showed the procedure which smoothes the chromatogram of FIG. 5 with the method of this invention. It is the figure which showed the system configuration used in the Example of this invention. a. The chromatogram for a verification in Example 1 is shown. b. The chromatogram which produced the level | step difference in about 7 minutes is shown. It is the figure which showed the procedure which smoothes the chromatogram of Example 1 with the method of this invention. a. The step part at the time of processing the chromatogram of Example 1 by a simple moving average method is shown. b. The peak 1 part at the time of processing the chromatogram of Example 1 by a simple moving average method is shown. a. The degree of influence reduction on the peak height by each treatment is shown. b. The influence on the peak half-value width by each processing is shown. In either case, the value before the processing is 100 and the fluctuation is shown. a. The chromatogram for a verification in Example 2 is shown. b. The chromatogram which produced the level | step difference in about 18.5 minutes is shown. It is the figure which showed the procedure which smoothes the chromatogram of Example 2 by the method of this invention. The step part at the time of processing the chromatogram of Example 2 by a simple moving average method is shown. a. The degree of influence reduction on the peak height by each treatment is shown. b. The influence on the peak half-value width by each processing is shown. In either case, the value before the processing is 100 and the fluctuation is shown. a. It is the figure which showed the peak detection in the unprocessed state when the level | step difference has arisen during the peak elution. b. It is the figure which showed the peak detection after the smoothing by a simple moving average method when the level | step difference has arisen during the peak elution. c. It is the figure which showed the peak detection after the smoothing by this invention when the level | step difference has arisen during the peak elution.

  Next, an Example is shown and this invention is demonstrated concretely. However, the present invention is not limited to these examples, and there is no problem even if the computer is connected to the chromatogram system and is operated according to a program.

  Using the liquid chromatogram system shown in FIG. 7, the actual measurement was performed, and after obtaining the raw chromatogram, the step processing of the present invention was performed. The system includes a solvent degassing device (SD-8020) 2, a liquid feed pump (DP-8020) 3, a sample injection device (AS-8020) 4, a column oven (CO-8020) 6, an ultraviolet-visible detector (UV- 8020) 7 and data processing device (LC-8020II) 9 (both manufactured by Tosoh Corporation).

As the analytical column 5, TSKgel ODS-100Z (5 μm, 4.6 mm ID × 15 cm) manufactured by Tosoh Corporation, eluent 1 is CH3CN / H2O = 50/50, and separation of p-hydroxybenzoic acids is performed. went. Other conditions are as follows Sample: p-Hydroxybenzoic Acid, Methyl p-Hydroxybenzoate, Ethyl p-Hydroxybenzoate, Propyl p-Hydroxybenzoate, Butyl p-Hydroxybenzoate, Hexyl p-Hydroxybenzoate, Heptyl p-Hydroxybenzoate each 8 [mu] g / 1 mL
Injection volume: 30 μL, column temperature: 40 ° C., flow rate: 1.0 mL / min
Data collection sampling pitch: 50 msec.

Example 1
First, after obtaining a normal chromatogram under the measurement conditions, operations 1) to 3) were performed to obtain a chromatogram in which a step was intentionally generated on the chromatogram. As a result, a chromatogram having a level difference of about 10 mABU is obtained about 7 minutes after sample injection.
1) Set the baseline position to 0_mABU before starting measurement (auto zero execution).
2) Start measurement (sample injection / data collection start).
3) After about 7 minutes, the baseline position is set to 10 mABU (automatic balance execution).

  FIG. 8a shows a normal chromatogram, and FIG. 8b shows a chromatogram in which a step is generated by the above operation. According to FIG. 8b, it can be seen that a step of about 10 mABU occurs in about 7 minutes.

  The chromatogram thus obtained was processed according to the following procedure. In the series of processes, chromatogram data obtained by the data processor LC-8020II was converted into text data, and various calculations were performed using commercially available spreadsheet software.

(Step 1)
A differential Δf / Sp was calculated for the acquired data (Sp: sampling pitch). The differential curve obtained in this way is shown in FIG. The fluctuation having the maximum and minimum points occurring in 7.3 to 7.8 minutes is a differential curve derived from the peak 4 like a normal distribution function. The difference in the baseline becomes a spike noise-like differential curve (about 7 minutes).

(Step 2)
Spike noise processing was performed using a “50% percentile filter”. The fluctuation having the maximum and minimum points occurring in 7.3 to 7.8 minutes is a differential curve derived from the peak 4 like a normal distribution function. It can be seen that the spike noise-like signal generated in about 7 minutes can be removed.

(Step 3)
The minute time interval area was calculated for the differential curve subjected to spike noise processing, and the cumulative total of the minute time interval areas was calculated. By this operation, the chromatogram can be reconstructed. It can be seen that the level difference that occurred in about 7 minutes has been eliminated by the series of operations described above.

(Comparative Example 1)
An untreated chromatogram similar to that of Example 1 was smoothed by the simple moving average method at 5 points, 11 points, 21 points, 51 points, and 101 points to be used, as shown in FIG. 10a. The general “simple moving average method” cannot solve the sudden base fluctuation (step) that occurs at about 7.00 minutes. Further, as shown in FIG. 10b, the portion of peak 1 has been affected by smoothing.

  FIG. 11a shows% height relative to the height of peak 1 of the untreated chromatogram, and FIG. 11b shows peak half-value width% similarly. It can be seen that the method of the present invention has little influence on both the peak height and the peak half-value width.

(Example 2)
First, after obtaining a normal chromatogram under the measurement conditions (the same conditions as in Example 1), the operations of 1) to 3) are performed to obtain a chromatogram in which a step is intentionally generated on the chromatogram. It was. As a result, a chromatogram having a level difference of about 80 mABU is obtained about 18.5 minutes after sample injection.
1) Set the baseline position to 0_mABU before starting measurement (auto zero execution).
2) Start measurement (sample injection / data collection start).
3) The baseline position is set to 10_mABU after about 18.5 minutes (auto zero execution).

  FIG. 12a shows a normal chromatogram, and FIG. 12b shows a chromatogram in which a step is generated by the above operation. Further, according to FIG. 12b, it can be seen that a step of about 80 mABU occurs while peak 5 is being eluted. The chromatogram thus obtained was treated in the same manner as in Example 1.

(Step 1)
A differential Δf / Sp was calculated for the acquired data (Sp: sampling pitch). The differential curve thus obtained is shown in FIG. The fluctuation having the maximum and minimum points occurring in 18.0 to 19.20 minutes is a differential curve derived from the peak 5 like a normal distribution function. The step in the peak becomes a spike noise-like differential curve (about 18.5 minutes).

(Step 2)
Spike noise processing was performed using a “50% percentile filter”. The fluctuation having the maximum and minimum points occurring in 18.0 to 19.20 minutes is a differential curve derived from the peak 5 like a normal distribution function. It can be seen that the spike noise-like signal generated at about 18.5 minutes can be removed.

(Step 3)
The minute time interval area was calculated for the differential curve subjected to spike noise processing, and the cumulative total of the minute time interval areas was calculated. By this operation, the chromatogram can be reconstructed. It can be seen that the level difference that occurred at about 18.5 minutes has been eliminated by the series of operations described above.

(Comparative Example 2)
An untreated chromatogram similar to that of Example 2 was smoothed by the “simple moving average method” at 5 points, 11 points, 21 points, 51 points, and 101 points to be used, as shown in FIG. As described above, the general “simple moving average method” cannot solve the sudden base fluctuation (step) generated at about 19.00 minutes.

  FIG. 15a shows% height relative to the height of peak 5 of the untreated chromatogram, and FIG. 15b shows peak half-value width% similarly. In the simple moving average method, regardless of the number of data points, the area of the peak 5 is extremely large and the peak half width is extremely small. This is because, as shown in FIG. 16a, the base line for peak 5 is greatly negatively inclined, the peak area is calculated to be abnormally high, and the half-value width is calculated to be abnormally small. On the other hand, it can be seen that the method of the present invention has little effect on both the peak height and the peak half-value width.

1. 1. Eluent 2. Deaeration device Liquid feed pump (sample side)
4). 4. Sample injection valve Analytical column 6. Column constant temperature bath UV-visible detector 8. Waste liquid9. System control and data processing equipment

Claims (3)

  Obtain a differential curve of the chromatogram, eliminate the differential signal change that has only one of the maximum value or the minimum value by spike noise processing, and integrate the obtained differential curve in a minute time section to obtain a chromatogram. A method for smoothing a chromatogram, comprising performing re-conversion.   The method for smoothing a chromatogram according to claim 1, wherein only a differential curve in a time domain in which a step of the chromatogram is generated is targeted.   The chromatogram smoothing method according to claim 1 or 2, wherein the chromatogram is a chromatogram obtained from liquid chromatography.

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