JP7215146B2 - Absorption photometric detector - Google Patents

Description

The present invention relates to a detector that uses an LED as a light source and is capable of outputting light in a plurality of wavelength ranges.

When using an absorptiometry detector for liquid chromatography, measurement is due to noise generated when switching the eluent, noise generated due to pulsation of the liquid feed pump, electrical noise, or undulation or drift of the baseline. In order to suppress the influence on the accuracy, a method of detecting the analytical component with the measurement wavelength light and using the reference wavelength light at the same time to correct the deviation of the measurement value is adopted. Generally, the measurement wavelength is set near the absorption maximum wavelength of the measurement component, and the reference wavelength is often set at a distance of 100 nm or more from the measurement wavelength so as not to be affected by the absorption spectrum of the measurement component.

When used as a light source for an absorptiometry detector, LEDs have been widely used recently because they have features such as longer life, higher luminous efficiency, and lower heat generation than halogen lamps and deuterium lamps. However, when a monochromatic LED is used alone, the emission wavelength band is extremely narrow, so if the measurement wavelength is set near the peak wavelength where the amount of light is high, it is possible to set the reference wavelength at an interval of 100 nm or more from the measurement wavelength. I have a problem that I can't.

Patent Literature 1 discloses a detector that outputs an LED for a primary wavelength and an LED for a secondary wavelength, which are capable of pulse output, at different timings when measuring using two wavelengths using an LED as a light source. However, when a plurality of LEDs with different output wavelengths are used, there is a risk that the ratio of the amount of light at the measurement wavelength and the amount of light at the reference wavelength will differ, resulting in different measurement results even when the same sample is measured.

Patent Literature 2 discloses a detector that uses a white light-emitting element that combines a blue LED and a special phosphor to have a sufficient emission intensity spectrum as one light source, and performs photometry at different detection units upstream and downstream of the flow cell. . However, if one of the detectors measures light using the measurement wavelength and the other detects light using the reference wavelength, the state of the sample changes while passing through the two detectors. There is a problem that the canceling effect of absorbance due to wavelength is reduced.

WO2014-157282 JP-A-11-304696

An object of the present invention is to provide a highly sensitive and highly accurate absorptiometry detector.

The present invention, which has been made to solve the above problems, includes the following inventions:
That is, the first aspect of the present invention is
a light-emitting portion having an LED device as a single light-emitting source;
a light-transmissive flow cell holding a measurement sample;
The light emitted by the light emitting unit is guided to the flow cell, and the light transmitted through the flow cell enters the light splitting element, and one of the two split lights is selectively output as light of a first wavelength. a light receiving section 1 having a light receiving element 1 that receives light through the element 1;
a light-receiving unit 2 having a light-receiving element 2 that receives the other of the two split lights through a wavelength-selecting element 2 that selectively outputs light of a second wavelength;
a calculation unit that converts the measured value of the measurement component in the measurement sample based on the transmitted light amount 1 of the first wavelength and the transmitted light amount 2 of the second wavelength;
An absorptiometry detector for a liquid chromatograph comprising
a light-emitting element in which the light-emitting unit emits light having a wavelength of at least 415 nm;
a phosphor that is excited by the light emitted by the light emitting element and emits light having a wavelength of any of 515 to 680 nm;
It is characterized by comprising:

Next, a second aspect of the present invention is
a light-emitting portion having an LED device as a single light-emitting source;
a light-transmissive flow cell holding a measurement sample;
The light emitted by the light emitting unit enters the light splitting element, and one of the two split lights is guided to the flow cell through the wavelength selection element 1 that selectively outputs the light of the first wavelength, and passes through the flow cell. a light receiving portion 1 having a light receiving element 1 for receiving the light received;
A light-receiving section 2 having a light-receiving element 2 that guides the other of the two split lights to an optical path cell through a wavelength selection element 2 that selectively outputs light of a second wavelength and receives the light that has passed through the optical path cell. and,
a calculation unit that converts the measured value of the measurement component in the measurement sample based on the transmitted light amount 1 of the first wavelength and the transmitted light amount 2 of the second wavelength;
An absorptiometry detector for a liquid chromatograph comprising
a light-emitting element in which the light-emitting unit emits light having a wavelength of at least 415 nm;
a phosphor that is excited by the light emitted by the light emitting element and emits light having a wavelength of any of 515 to 680 nm;
It is characterized by comprising:

Highly sensitive and accurate hemoglobin by setting the reference wavelength longer than the measurement wavelength by 100 nm or more using an absorptiometry detector equipped with a single LED device composed of a light emitting element and a phosphor as a light source. It becomes possible to measure

It is the figure which showed the one aspect|mode of the absorption photometric detector of this invention. FIG. 4 shows another embodiment of the absorptiometric detector of the present invention; 1 is a diagram showing the configuration of a liquid chromatograph incorporating the absorption photometric detector of the present invention; FIG. FIG. 3 is a diagram showing an emission spectrum of a light-emitting element coated with an aluminate-based yellow-green phosphor; FIG. 4 is a diagram showing a chromatogram obtained by measuring at a measurement wavelength of 415 nm and a reference wavelength of 600 nm using a light-emitting element coated with an aluminate-based yellow-green phosphor. FIG. 4 is a chromatogram showing the measurement wavelength, the reference wavelength and the level of noise drift after calculation; FIG. 2 is a diagram showing an absorption spectrum of hemoglobin in whole blood; FIG. 3 is a diagram showing an emission spectrum of a light-emitting element coated with a nitride-based orange phosphor; FIG. 4 is a diagram showing a chromatogram obtained by using a light-emitting element coated with a nitride-based orange phosphor and measuring at a measurement wavelength of 415 nm and a reference wavelength of 600 nm. FIG. 4 is a diagram showing a chromatogram obtained by measuring at a measurement wavelength of 415 nm and a reference wavelength of 650 nm using a light-emitting element coated with a nitride-based orange phosphor. FIG. 4 is a diagram showing an emission spectrum of a light-emitting element coated with a nitride-based red phosphor; FIG. 4 is a diagram showing a chromatogram obtained by using a light-emitting element coated with a nitride-based red phosphor and measuring at a measurement wavelength of 415 nm and a reference wavelength of 650 nm. FIG. 3 is a diagram showing an emission spectrum of a blue LED having broad characteristics; FIG. 4 is a diagram showing a chromatogram obtained by using a blue LED with broad characteristics and measuring at a measurement wavelength of 415 nm and a reference wavelength of 500 nm.

The present invention will be described in detail below.

An LED device is a light-emitting element that is molded using a method such as mixing a fluorescent substance into transparent silicon resin, transparent fluorine resin, transparent epoxy resin, etc., and coating the light-emitting element so as to surround the upper part of the light-emitting element, and then mounted in a package. and ramped. The shape is exemplified by general cannonball, hat, chip, flux, and the like.

The light emitting element refers to a semiconductor light emitting element (LED chip), and a violet to blue-violet light emitting element that emits light with a shorter wavelength than a blue light emitting element can be preferably used. In terms of wavelength, a light-emitting element with a peak wavelength of about 380 to 450 nm, preferably about 400 to 430 nm, is suitable because it can sufficiently ensure the amount of light detected at the maximum absorption wavelength of 415 nm for hemoglobins (oxyhemoglobins).

The phosphor is an aluminate-based, silicate-based, or YAG (yttrium aluminum garnet)-based green-emitting phosphor (peak wavelength of about 520 to 550 nm) or yellow-emitting phosphor (peak wavelength of about 550 to 580 nm), nitride An orange-emitting phosphor (peak wavelength of about 580 to 620 nm) or a red-emitting phosphor (peak wavelength of about 620 to 680 nm) is exemplified. The phosphor is excited by the light emitted from the light emitting element and emits light containing light with a wavelength of any of 515 to 680 nm.

The flow cell can be exemplified by those molded into a general cylindrical shape, rectangular parallelepiped shape, vertical table shape, or the like. Materials made of quartz glass, plastic, etc. can be used, and as long as at least the light emitted from the light source can pass through, it does not have complete transparency, even if it is partially transparent. good. The optical path cell is preferably similar in shape and material to the flow cell.

An optical branching element refers to an element that spectrally separates light by transmitting or reflecting light depending on the wavelength, and examples thereof include a dichroic mirror and a half mirror. For example, a long-pass dichroic mirror exhibits high reflectance for light with wavelengths shorter than the cutoff wavelength and exhibits high transmittance for light with wavelengths longer than the cutoff wavelength. A short-pass dichroic mirror has high transmittance for light with wavelengths shorter than the cutoff wavelength and high reflectance for light with wavelengths longer than the cutoff wavelength.

The wavelength selection element is exemplified by a bandpass filter (interference filter) having a spectral half width of about 5 to 20 nm, which selectively transmits only light in a specific wavelength band.

A light-receiving element refers to a general semiconductor photodetector that converts light into electricity, and is exemplified by a photodiode, a phototransistor, and the like. A light-receiving part refers to a printed circuit board on which a light-receiving element is mounted.

The calculation unit acquires an analog signal output from the light receiving unit, converts it into a digital signal, analyzes the converted value, converts it into a chromatogram, converts it into a measured value, and outputs it. For example, in the case of two-wavelength measurement, the output voltage obtained from the light-receiving part of the measurement wavelength light, the output voltage obtained from the light-receiving part of the reference wavelength light, and the ratio of these output voltages are logarithmically transformed, and the converted value is After analysis, it is converted into a chromatogram and converted into a measured value and output.

The optical system of the present invention can be exemplified by the following configurations.

As one aspect (optical system 1), as shown in FIG. A flow cell 103 holding a measurement sample, a long-pass dichroic mirror 104 for splitting the light passing through the flow cell 103 into two, a band-pass filter 105 for obtaining measurement wavelength light from one of the split lights, and receiving measurement wavelength light. A light receiving section 106 having a light receiving element for detection, a bandpass filter 107 for obtaining reference wavelength light from the other branched light, a light receiving section 108 having a light receiving element for receiving and detecting the reference wavelength light, a light receiving section 106 and a light receiving section 108. , and converts it into the measured value of the measurement component in the measurement sample.

As another aspect (optical system 2), as shown in FIG. A long-pass dichroic mirror 204 for branching, a band-pass filter 207 for obtaining measurement wavelength light from one of the branched lights, a flow cell 203 for receiving the light that has passed through the band-pass filter 207, and receiving and detecting the light that has passed through the flow cell 203. a light-receiving unit 208 having a light-receiving element for light reception, a band-pass filter 205 for obtaining reference wavelength light from the other branched light, a reference optical path cell 210 for inputting the light that has passed through the band-pass filter 205, and a reference optical path cell 210. It is composed of a light receiving section 206 having a light receiving element for detecting light that has passed through, and a computing section 209 for inputting signals output from the light receiving sections 206 and 208 and converting them into measured values of measurement components in the measurement sample.

The liquid chromatograph equipped with the absorptiometry detector of the present invention configured by the above-described optical system 1 or optical system 2 uses the first wavelength of 415 nm output by the wavelength selection element 1 as the measurement wavelength, and the wavelength selection element 2 By using the output second wavelength of any one of 515 to 680 nm, preferably any one of 600 to 650 nm as a reference wavelength, hemoglobins can be suitably measured.

EXAMPLES The present invention will be described in more detail below using examples, etc., but the aspects of the present invention are not limited to these.

FIG. 3 is a schematic diagram of a liquid chromatograph used when confirming the effects of the present invention.
The eluents are Liquid 1 (G9 eluent HSi 1st liquid Tosoh Corporation) 301, Liquid 2 (G9 eluent HSi 2nd liquid Tosoh Corporation) 302 and Liquid 3 (G9 eluent HSi 3rd liquid Tosoh Corporation). 303 was used.
Each eluent was sent at a flow rate of 2.0 mL/min using a liquid sending pump (DP-8020 Tosoh Corporation) 306. Each eluent was degassed through a deaerator 304, and step gradient elution was performed by switching electromagnetic valves (305a, 305b, 305c) in the order of liquid 3, liquid 2, liquid 1. In order to measure the stable glycosylated hemoglobin in blood, which is the object to be measured, an autosampler (AS-8020, Tosoh Corporation) 307 including an injection valve was used to inject a 200-fold diluted sample of healthy human blood. Sample injection used a 5 μL sample loop. A sample was injected into a column 308 filled with a cation exchange resin TSKgel SP-NPR (Tosoh Corporation) as a packing material for adsorbing and desorbing hemoglobin components in blood, and each separated hemoglobin component was analyzed using the optical system shown in FIG. Detected by an absorptiometry detector 309 having a system (the shape of the flow cell is a cylinder with an inner diameter of 1.0 mm and an optical path length of 10 mm). As the signal output from the detector, the output voltage Vsam obtained from the light of the measurement wavelength, the output voltage Vref obtained from the light of the reference wavelength, and the ratio of the output voltage obtained from the light of the measurement wavelength to the output voltage obtained from the light of the reference wavelength. A logarithmically converted converted output Vout (log(Vref/Vsam)) was obtained. Analog signals of Vsam, Vref, and Vout are acquired by a controller equipped with LC-8020 (Tosoh Corporation), and output value analysis, drawing, data processing and control of each unit are performed by LC-8020model2 (Tosoh Corporation). rice field.

(Example 1)
Using an LED device in which the upper part of the blue-violet light emitting element (peak wavelength about 430 nm) is molded with a transparent silicone resin containing an aluminate-based yellow-green phosphor (peak wavelength about 550 nm), the output wavelength of the bandpass filter for the measurement wavelength was set to 415 nm, and the output wavelength of the band-pass filter for the reference wavelength was set to 600 nm, and the stable glycated hemoglobin in whole blood was measured. FIG. 4 shows the emission spectrum of the LED device, and FIG. 5 shows the chromatogram of Vsam, Vref, and Vout obtained by the detector.
The hemoglobin A0 peak detected using the output Vref obtained from the reference wavelength light is 1.33% of the hemoglobin A0 peak detected using the output Vsam obtained from the measurement wavelength light, and the measurement on the reference wavelength side is small. I was able to confirm that. In addition, it was confirmed from the Vout chromatogram that an unstable hemoglobin A1c peak (401), a stable hemoglobin A1c peak (402), and a hemoglobin A0 peak (403) were obtained.

In order to achieve high sensitivity and high precision measurement, it is essential to reduce baseline drift and noise. As an example showing the merit of setting the reference wavelength to be longer than the measurement wavelength by 100 nm or more, noise and drift were evaluated by switching the eluent from liquid 3 to liquid 2 and then liquid 1 in the order of liquid 3, liquid 2, and liquid 1 under the above detection conditions, and Vsam , Vref, and Vout are shown in FIG. Vsam was 0.08 mV drift, 0.073 mV maximum noise, 0.17 mV switching noise, and Vref was 0.08 mV drift, 0.067 mV maximum noise, 0.31 mV switching noise. On the other hand, Vout has a drift of 0.01 mV, a maximum noise of 0.011 mV, and a switching noise of 0.03 mV. It is possible to reduce switching noise by 82%, achieving highly sensitive and highly accurate measurements.

Furthermore, in order to demonstrate the merit of using a reference wavelength of 600 nm or more for measuring hemoglobins in whole blood, the absorption spectrum of hemoglobin in whole blood was measured using a spectrophotometer (UV-2600, Shimadzu Corporation). is shown in FIG. In the obtained absorption spectrum, the wavelength range centered at about 415 nm and the wavelength range from 520 nm to 600 nm show absorption specific to hemoglobin, and no significant absorption is shown at 600 nm or higher. By setting it, the influence of the hemoglobin concentration is reduced, and it is possible to measure with less measurement on the reference wavelength side and with higher accuracy.

(Example 2)
Stable glycated hemoglobin in whole blood was measured under the same conditions as in Example 1, except that the phosphor was changed to a nitride-based orange phosphor (peak wavelength: about 620 nm). FIG. 8 shows the emission spectrum of the LED device, and FIG. 9 shows the chromatogram of Vsam, Vref, and Vout obtained by the detector.
The hemoglobin A0 peak detected using the output Vref obtained from the reference wavelength light is 1.37% of the hemoglobin A0 peak detected using the output Vsam obtained from the measurement wavelength light, and the measurement on the reference wavelength side is small. I was able to confirm that. In addition, it was confirmed from the Vout chromatogram that an unstable hemoglobin A1c peak (401), a stable hemoglobin A1c peak (402), and a hemoglobin A0 peak (403) were obtained.

(Example 3)
Stable glycated hemoglobin in whole blood was measured under the same conditions as in Example 2, except that the output wavelength of the bandpass filter for measurement wavelength was changed to 415 nm and the output wavelength of the bandpass filter for reference wavelength was changed to 650 nm. FIG. 10 shows chromatograms of Vsam, Vref and Vout obtained by the detector.
The hemoglobin A0 peak detected using the output Vref obtained from the reference wavelength light is 0.12% of the hemoglobin A0 peak detected using the output Vsam obtained from the measurement wavelength light, and the measurement on the reference wavelength side is small. I was able to confirm that. In addition, it was confirmed from the Vout chromatogram that an unstable hemoglobin A1c peak (401), a stable hemoglobin A1c peak (402), and a hemoglobin A0 peak (403) were obtained.

(Example 4)
Stable glycated hemoglobin in whole blood was measured under the same conditions as in Example 3, except that the phosphor was changed to a nitride-based red phosphor (peak wavelength: about 660 nm). FIG. 11 shows the emission spectrum of the LED device, and FIG. 12 shows the chromatogram of Vsam, Vref, and Vout obtained by the detector.
The hemoglobin A0 peak detected using the output Vref obtained from the reference wavelength light is 0.15% of the hemoglobin A0 peak detected using the output Vsam obtained from the measurement wavelength light, and the measurement on the reference wavelength side is small. I was able to confirm that. In addition, it was confirmed from the Vout chromatogram that an unstable hemoglobin A1c peak (401), a stable hemoglobin A1c peak (402), and a hemoglobin A0 peak (403) were obtained.

(Comparative example 1)
In Comparative Example 1, an LED device composed of a blue light-emitting element having broadband characteristics and containing no phosphor was used as the light source. FIG. 13 shows the emission spectrum of this LED device.
Although the LED device has a central wavelength of about 460 nm, it was found that it hardly emits light in the wavelength band after 560 nm. Therefore, with this light source, it was impossible to set a wavelength of 560 nm or more as the reference wavelength. The output wavelength of the band-pass filter used 415 nm as the measurement wavelength and 500 nm as the reference wavelength. Stable glycated hemoglobin in whole blood was measured using an absorptiometry detector equipped with this LED device. FIG. 14 shows chromatograms of Vsam, Vref, and Vout obtained by the detector.
The hemoglobin A0 peak detected using the output Vref obtained from the reference wavelength light was 13.66% of the hemoglobin A0 peak detected using the output Vsam obtained from the measurement wavelength light, compared with Examples 1 to 4. It was confirmed that the measurement on the reference wavelength side is large. In addition, it was confirmed from the Vout chromatogram that an unstable hemoglobin A1c peak (401), a stable hemoglobin A1c peak (402), and a hemoglobin A0 peak (403) were obtained.

101, 201: LED devices 102, 202: collimating lenses 103, 203: flow cells 104, 204: dichroic mirrors 105, 107, 205, 207: bandpass filters 106, 108, 206, 208: light receiving units 109, 209: computing units 210: optical path cell for reference 301: liquid 1 302: liquid 2 303: liquid 3 304: deaerator 305a, 305b, 305c: solenoid valve 306: liquid feed pump 307: autosampler 308: column 309: absorbance detector 401 : unstable hemoglobin A1c peak 402: stable hemoglobin A1c peak 403: hemoglobin A0 peak

Claims (4)

a light-emitting portion having an LED device as a single light-emitting source;
a light-transmissive flow cell holding a measurement sample;
The light emitted by the light emitting unit is guided to the flow cell, and the light transmitted through the flow cell enters the light splitting element, and one of the two split lights is selectively output as light of a first wavelength. a light receiving section 1 having a light receiving element 1 that receives light through the element 1;
a light-receiving unit 2 having a light-receiving element 2 that receives the other of the two split lights through a wavelength-selecting element 2 that selectively outputs light of a second wavelength;
a calculation unit that converts the measured value of the measurement component in the measurement sample based on the transmitted light amount 1 of the first wavelength and the transmitted light amount 2 of the second wavelength;
An absorptiometry detector for a liquid chromatograph comprising
a light-emitting element in which the light-emitting unit emits light having a wavelength of at least 415 nm;
Excited by the light emitted by the light-emitting element , a green-emitting phosphor (peak wavelength of about 520-550 nm), a yellow-emitting phosphor (peak wavelength of about 550-580 nm), an orange-emitting phosphor (peak wavelength of about 580-620 nm) about) or a red-emitting phosphor (with a peak wavelength of about 620-680 nm) ; and
The absorptiometry detector, comprising: a light-emitting portion having an LED device as a single light-emitting source;
a light-transmissive flow cell holding a measurement sample;
The light emitted by the light emitting unit enters the light splitting element, and one of the two split lights is guided to the flow cell through the wavelength selection element 1 that selectively outputs the light of the first wavelength, and passes through the flow cell. a light receiving portion 1 having a light receiving element 1 for receiving the light received;
A light-receiving section 2 having a light-receiving element 2 that guides the other of the two split lights to an optical path cell through a wavelength selection element 2 that selectively outputs light of a second wavelength and receives the light that has passed through the optical path cell. and,
a calculation unit that converts the measured value of the measurement component in the measurement sample based on the transmitted light amount 1 of the first wavelength and the transmitted light amount 2 of the second wavelength;
An absorptiometry detector for a liquid chromatograph comprising
a light-emitting element in which the light-emitting unit emits light having a wavelength of at least 415 nm;
a phosphor that is excited by the light emitted by the light emitting element and emits light having a wavelength of any of 515 to 680 nm;
The absorptiometry detector, comprising: A method for measuring hemoglobins by a liquid chromatograph equipped with the absorptiometry detector according to claim 1 or 2,
The above method, wherein the first wavelength of 415 nm output by the wavelength selection element 1 is used as a measurement wavelength, and the second wavelength of any one of 515 to 680 nm output by the wavelength selection element 2 is used as a reference wavelength. A method for measuring hemoglobins by a liquid chromatograph equipped with the absorptiometry detector according to claim 1 or 2,
The above method, wherein the first wavelength of 415 nm output by the wavelength selection element 1 is used as a measurement wavelength, and the second wavelength of 600 to 650 nm output by the wavelength selection element 2 is used as a reference wavelength.

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