JPH05142143A - Spectrum collecting method for detector of spectrophotometer - Google Patents

Abstract

PURPOSE:To obtain the title method by which a spectrum can be accurately collected even in an on-flow state. CONSTITUTION:After emitting light having a wavelength lambda0 and monitoring the output of an optical sensor 8 in an on-flow state, the rotation of a grating 7 is started at appropriate timing tA. By rotating the grating 7 and successively storing the output signal of the sensor 8 in a memory 12, scanning is performed with the emitted light until the wavelength of the light changes from lambda1 to lambda2 and, after scanning, the wavelength is returned to lambda0. Then the error of the measured data stored in the memory 12 associated with the concentration change of a sample to be measured is removed by correcting the data by utilizing the scan starting time tA scan terminating time tB, and the levels PA an PB of the output signal against the wavelength lambda0 immediately before starting and immediately after terminating the scanning.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for collecting a spectrum in a liquid chromatograph, a supercritical chromatograph, a capillary electrophoresis apparatus and the like.

[0002]

2. Description of the Related Art Chromatograph spectrophotometer detectors include
For example, there is an absorption detector shown in FIG. This absorption detector is composed of an optical system 1 for irradiating a flow cell with inspection light, and an electric system 2 for detecting absorbance. The optical system 1 is
The light source 3, the slit 4, the mirrors 5 and 6, the grating 7, and the beam splitter 14 for separating the sample light and the reference light. Further, the electric system 2 includes an optical sensor 8 _a that converts transmitted light into an electric signal, an amplifier 9 _a that amplifies the output of the optical sensor 8 _a , and an A / D converter that converts the output of the amplifier 9 _a into a digital signal. 10 _a , an optical sensor 8 _b for converting the reference light into an electric signal, an amplifier 9 _b for amplifying the output of the optical sensor 8 _b , and an A / D converter 10 _b for converting the output of the amplifier 9 _b into a digital signal. It is composed of a CPU 11 that appropriately processes the captured digital signal, and a memory 12 that stores a program and the like.

In the absorption detector having the above structure,
For example, the wavelength λ ₀Irradiate the inspection light of
The transmitted light level and the reference light level.
Service 8_aAnd 8_bIf time-sequential detection is performed by
The time transition can be obtained. Also, the wavelength of the irradiation light
If you want to measure the absorbance spectrum by changing
Stop the flow in the flow cell by some method,
In that state, while rotating the grating 7,
Sensors 8a and 8_bDetect the output level of. Usually move
The wavelength scan is performed with the phase filled in the cell, and the
The background spectrum is collected and stored. Then try
Fill the cell with the sample solution and perform a spectrum scan to obtain the
The background spectrum is subtracted from the
And get the true spectrum.

[0004]

However, in the above-mentioned conventional apparatus, it is impossible to measure the spectrum such as the absorbance in the on-flow state, and the wavelength of the inspection light is scanned by temporarily rotating the grating. However, there is a problem that a true spectrum cannot be collected. Because,
When a spectrum is sampled by scanning the wavelength of the inspection light, the concentration of the sample to be measured changes within the scanning time, so the sampled spectrum deviates from the true value, and this deviation is the rate of concentration change. This is because there is no reproducibility because it changes depending on.

The present invention has been made in view of this problem, and an object thereof is to provide a spectrum sampling method for a spectrophotometer detector capable of collecting an accurate spectrum even in an on-flow state.

[0006]

In order to achieve the above object, a spectrum sampling method for a spectrophotometer detector according to a first aspect of the present invention is such that a wavelength of irradiation light is scanned in an on-flow state in a spectrophotometer detector. Method to collect the spectrum of the sample by irradiating the sample to be measured with light of a specific wavelength in the on-flow state, and monitoring the output signal level of transmitted light and fluorescence in this state. When a predetermined condition is established by the transition of, while starting the scanning of the wavelength of the irradiation light, while scanning the wavelength of the irradiation light within a predetermined range,
The output signal data is stored in the storage device, and after the scanning is completed, the wavelength is returned to the value before the start of the scanning, and the measurement data stored in the storage device is set to the scanning start time, the scanning end time, and the wavelength of the wavelength. And at least two sets of the level of the output signal with respect to the specific wavelength at the time when the specific wavelength is reached by scanning and immediately before the start of scanning, immediately after the end of scanning, and at the time when the specific wavelength is reached by scanning. Then, an error amount due to a change in the concentration of the sample to be measured is corrected.

Further, a spectrum sampling method for a spectrophotometer detector according to a second aspect is the method according to the first aspect,
Recognizing the peak rising point and peak maximum point of the output signal, after recognizing this peak maximum point, wavelength scanning is started at a predetermined timing obtained from the difference between the peak maximum point and peak rising point. And

[0008]

The spectrophotometer detector irradiates the flow cell with light of a specific wavelength λ _{0 in} the on-flow state, monitors the level of transmitted light, fluorescence, etc. in this state, and determines a predetermined value based on the transition of the output signal level. When the condition is satisfied, the scanning of the wavelength of the irradiation light is started. For example, when the level of the output signal falls below the peak value by a predetermined value (time t _A ), scanning of irradiation light is started.

After the scanning of the irradiation light is started, the wavelength of the irradiation light is changed.
Within a predetermined range (eg λ₁~ Λ₂) And then
(Time t_B), The wavelength λ before the start of scanning₀Return to.
During the scanning of the irradiation light, the output signal data is
Data in a storage device. When the above process is completed,
The wavelength of the measurement data stored in the storage device
Scan start time (t_A) And scan end time (t_B) And wavelength
The time (t_c), And
And immediately before the start of scanning and immediately after the end of scanning
Output signal for the specific wavelength at the time when the wavelength is reached
Issue level P _A, P_B, P_cAt least two sets of
Use it to correct the error caused by the change in the concentration of the sample to be measured.
It By this correction process, the measured
Since the effect of changes in concentration is eliminated, the
The accurate spectrum can be collected even if it is.

The present spectrum sampling method may be implemented as a spectrophotometer detector alone, or may be implemented by an integrator or a personal computer data processing unit that processes a signal from the spectrophotometer detector. Further, it is the same as the conventional method that the background spectrum is first detected and the background spectrum is subtracted from the spectrum collected when the sample passes through the cell.

[0011]

EXAMPLES A spectrophotometer detector for realizing the present invention will be described in detail below based on examples. FIG. 1 is a schematic view of an absorption detector which is an embodiment for realizing the present invention. The hardware configuration of this device is almost the same as that of the conventional one, and includes a light source 3, a slit 4, mirrors 5 and 6, a grating 7, a beam splitter 14, and optical sensors 8 _a and 8 _b . and the amplifier 9 _a, 9 _b,
It is composed of A / D converters 10 _a and 10 _b , a CPU 11, and a memory 12.

Here, the grating 7 is configured to rotate as needed by a drive mechanism (not shown). Then, when the grating 7 is in a stationary state, the flow cell 13 is irradiated with light having a wavelength λ ₀ , and when the grating 7 rotates, the wavelength of the irradiation light is correspondingly λ ₁
To λ _{2 are} scanned. FIG. 2 shows an example of how the absorbance of the sample component changes with time with respect to the irradiation light of the wavelength λ _{0. Since} the sample component is moving, the absorbance is the peak rising edge. It starts to increase from the point T ₀ and gradually decreases when it exceeds the peak top point T _P.

The operation of the apparatus shown in FIG. 1 will be described below with reference to FIG. First, the grating 7 is kept stationary, and the flow cell 13 is irradiated with light having a wavelength λ ₀ . C
PU11 is an output of the optical sensor 8 _a and 8 _b monitors in this state, the level is a predetermined level of the output signal, for example, wait for reaching the position of the point A in FIG. When the signal level reaches the position of point A in FIG. 2, the driving mechanism (not shown) rotates the grating 7 to scan the wavelength of the irradiation light from λ ₁ to λ ₂ . Further, the CPU 11 fetches the outputs of the optical sensors 8 _a and 8 _b for each wavelength and sequentially stores them in the memory 12. After scanning the wavelength up to λ ₂ , the wavelength is returned to the initial value λ ₀ (see point B in FIG. 2). That is, the broken line portion of FIGS.
_The time required for scanning is shown from ₁ to λ ₂ , and it shows that the spectrum acquisition was started at the time point A in FIG. 2 and was completed at the time point B.

S _{1 in} FIG. 4 shows the measurement result when the absorbance is measured by changing the wavelength from λ ₁ to λ ₂ . As described above, the wavelength changes from λ ₁ to λ ₂ from time A to time B. By the way, since the above measurement is carried out in the on-flow state, from the measurement of the absorbance for the wavelength λ ₁ to the measurement of the absorbance for the wavelength λ ₂ , the concentration of the sample component gradually decreases, and the measurement The resulting curve S ₁ will deviate from the correct spectrum by this decrease in concentration. S _{0 in} FIG. 4 is A
FIG. 7 is a diagram showing a true spectrum at a time point, and comparing this true spectrum S ₀ with the measurement result S ₁ shows that they match at time A (wavelength λ ₁ ) but at time B (wavelength λ ₂ ). It will be quite different. Since the difference between the correct spectrum S ₀ and the measurement result S ₁ differs depending on the changing speed of the concentration, it depends on the time required for scanning the wavelength and the height of the peak and lacks reproducibility.

Therefore, the CPU 11 next proceeds to the memory 12
The measurement data S ₁ (λ) stored inside is corrected by the following procedure. Note that, here, the primary correction is performed on the assumption that the density change and the like from time A to time B are all linear. (I) The time t (λ) when the light having the wavelength λ is irradiated is obtained from the equation. t (λ) = t _A + (t _B −t _A ) × (λ−λ ₁ ) / (λ
₂₋ λ ₁ ) ... Note that t _A and t _B are the time points A and B ((b) in FIG. 2).
P _A and P _B are the levels of absorbance at points A and B (see FIG. 2B) λ ₁ and λ ₂ are wavelengths at points A and B. (Ii) The absorbance P (t (λ)) for the wavelength λ _{0 at the} time t (λ) when the inspection light of the wavelength λ is irradiated is obtained from the formula. P (t (λ)) = P A - (P A -P B) × (t (λ) -
t _A ) / (t _B −t _A ) ... (iii) Using the absorbance P _A at time _A and the absorbance P (t (λ)) at time t (λ) for wavelength λ ₀ ,
The measurement result S ₁ (λ) for the wavelength λ is corrected using the formula. S (λ) = S ₁ (λ) × P _A / P (t (λ)) ... The calculation results (i) to (iii) can eliminate the influence of the change in concentration. S ₁ (λ)
Will be corrected to the true spectrum S ₀ (λ).

In the above description, the starting point of wavelength scanning (point A in FIG. 2) was not particularly limited, but if the wavelength was scanned before and after the peak apex, effective data could be obtained. Absent. Therefore, the CPU 11
The scanning start point is recognized by the following method (hereinafter, refer to FIG. 3). That is, the CPU 11 causes the peak rising point T _0.
And the peak top point T _P are recognized and their time interval t _k
(= T _P -T ₀₎ was calculated, after recognizing the peak top point T _P, it begins scanning wavelengths than time, for example t _k / 3 elapsed. In this way, when the scanning start point is determined,
The amount of change in absorbance or the like with respect to the change in concentration becomes almost linear, which is preferable as a measurement environment.

In this embodiment, the primary correction is made on the assumption that the density change between points A and B is linear, but the output values P _A and P _B at points _A and _B are changed. other,
Output value P for light of wavelength λ ₀ in the wavelength scanning process
Higher-order correction may be performed using _C (see point _{C in} FIG. 4). Further, the peak waveform may be fitted to the Gaussian distribution for correction.

As described above, the absorption detector has been described in this embodiment, but the present invention is not limited to this, and is similarly applicable to, for example, a fluorescence spectrophotometer.

[0019]

As described above, in the spectrophotometer detector spectrum sampling method according to the present invention, this data is appropriately corrected after scanning the wavelength of the irradiation light to sample the spectrum. That is, even if the concentration of the sample to be measured changes, its influence is removed, so that an accurate spectrum can be obtained even in the on-flow state.

[Brief description of drawings]

FIG. 1 illustrates a spectrophotometer detector which is an embodiment for implementing the present invention.

FIG. 2 is a graph showing the change with time of the absorbance with respect to a specific wavelength λ ₀ .

FIG. 3 is a diagram for explaining a scanning start point A of a wavelength.

FIG. 4 illustrates a true spectrum S ₀ and a measured spectrum S ₁ .

[Explanation of symbols]

 1 Optical system 2 Electric system 7 Grating 11 CPU 12 Memory

Claims (2)

[Claims] 1. A method of scanning a wavelength of irradiation light in an on-flow state to collect a spectrum of a sample, which comprises irradiating a sample to be measured with light of a specific wavelength in an on-flow state,
In this state, the output signal level of transmitted light, fluorescence, etc. is monitored, and when the predetermined condition is satisfied due to the transition of the output signal level, the scanning of the wavelength of the irradiation light is started and the irradiation light While scanning the wavelength, the output signal data is stored in the storage device, after the scanning is completed, the wavelength is returned to the value before the start of the scanning, and the scanning start time of the wavelength is measured for the measurement data stored in the storage device. And the scanning end time and the time when the wavelength is reached by scanning the wavelength, and the level of the output signal for the specific wavelength immediately before the start of scanning, immediately after the end of scanning, and at the time when the specific wavelength is reached by scanning, A spectrum sampling method for a spectrophotometer detector, characterized in that at least two sets are used to correct an error amount due to a change in concentration of a sample to be measured. 2. The invention according to claim 1, wherein the peak rising point and the peak maximum point of the output signal are recognized, the peak maximum point is recognized, and then a predetermined value is obtained from the difference between the peak maximum point and the peak rising point. A method for collecting a spectrum for a spectrophotometer detector, which starts scanning of a wavelength at the timing of.