JPH0712798A - Fluorescence detection device for liquid chromatograph - Google Patents

Abstract

PURPOSE:To provide a fluorescence detection device for a liquid chromatograph, ensuring detection with high sensitivity even in an ultraviolet zone CONSTITUTION:Light emitted from an illuminant 1 is applied on a beam splitter 5 through a condenser lens 2, a slit 3 and a diffraction grating 4. A part of the light is reflected for use as reference light and the optical intensity thereof is detected with an optical detecting element 6. On the other hand, the light through the beam splitter 5 is applied on a fluorescent sample in a cell 10 through the slit 3. The optical intensity of the fluorescence emitted from the sample is detected with an optical detecting element 14. Output from both elements 6 and 14 is sent to CPU 16 through preamplifiers 7 and 15, and an A/D converter 8, and the optical intensity of the fluorescence is divided with the optical intensity of the reference light, thereby calculating relative intensity. In a liquid chromatograph so constituted, an illuminant having high optical intensity in an ultraviolet zone is constituted of a xenon-mercury lamp where xenon and mercury are mixed and sealed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence detector for liquid chromatograph.

[0002]

2. Description of the Related Art The measurement wavelength range of a fluorescence detector for a liquid chromatograph is wide ranging from the ultraviolet range to the visible range.
Therefore, conventionally, a xenon lamp has been used as the light source of the detector. This is because the xenon lamp emits continuous light while maintaining the light intensity above a certain level from the ultraviolet range to the visible range. Further, the light intensity of the xenon lamp tends to increase as the wavelength becomes longer.

[0003]

By the way, the fluorescence detector for liquid chromatograph is required to measure with high sensitivity, and in recent years, the requirement is more increased. Then, as described above, since the light intensity of the xenon lamp in the ultraviolet region is low, it becomes impossible to emit the light intensity necessary for performing high-sensitivity measurement. Therefore, it is conceivable to secure the required light intensity in the ultraviolet region by using a xenon lamp with high power consumption and increasing the light intensity as a whole. However, in such a case, since heat generation also increases with an increase in power consumption, a heat dissipation mechanism is required, and the device becomes large. Furthermore, since the emission intensity in the visible range also increases,
The light in the visible range may cause stray light in the measurement, which may rather reduce the measurement sensitivity.

The present invention has been made in view of the above background, and an object of the present invention is to provide a fluorescence detection device for a liquid chromatograph capable of detecting with high sensitivity even in the ultraviolet region. .

[0005]

In order to achieve the above-mentioned object, in a fluorescence detection apparatus for a liquid chromatograph according to the present invention, a light source, a cell in which a fluorescent sample is installed, and light emitted from the light source are provided. An optical system for irradiating the fluorescent sample in the cell, a light intensity of the light before the irradiation, and a means for detecting the light intensity of the fluorescence emitted from the fluorescent sample in the cell, respectively, are detected by the detecting means. In the fluorescence detection device for a liquid chromatograph equipped with means for performing a predetermined calculation process based on both light intensities, the light source is composed of a xenon-mercury lamp in which xenon and mercury are mixed and sealed.

[0006]

The function of the xenon-mercury lamp is high in the ultraviolet region. Therefore, the light intensity of the excitation light with which the fluorescent sample in the cell is irradiated is also increased, and accordingly, the light intensity of the fluorescent light emitted from the fluorescent sample is also improved. Since the signal S / N is proportional to the square root of the light intensity, highly sensitive measurement can be performed.

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The fluorescence detection device for liquid chromatograph according to the present invention will be described in detail below with reference to the accompanying drawings. Figure 1
Shows a schematic configuration of a liquid chromatograph device to which the present invention is applied. As shown in the figure, a condenser lens 2, an excitation side entrance slit 3 is provided on the optical path of the light emitted from the light source 1.
Are arranged in this order so that the emitted light passes through the condenser lens 2 and is condensed on the entrance slit 3.
Then, the light passes through the entrance slit 3 and is irradiated onto the excitation light side concave diffraction grating 4, and the light is dispersed there.

Further, a beam splitter 5 is arranged on the optical path of the dispersed excitation light. Then, the excitation-side photodetection element 6 is arranged on the optical path of the excitation light that is irradiated to and reflected by the beam splitter 5. This excitation side light detection element 6
For example, a photomultiplier tube or the like is used, and the light intensity is converted into an electric signal. The output of the excitation-side photodetection element 6 is connected to the preamplifier 7, and the output of the preamplifier 7 is connected to the A / D converter 8.

On the other hand, an exit slit 9 and a cell 10 are arranged in that order on the optical path of the excitation light transmitted through the beam splitter 5. As a result, the excitation light passes through the exit slit 9 and is applied to the fluorescent sample in the cell 10.
Further, the fluorescence-side incident slit 11 is arranged at a predetermined position in the emission direction of the fluorescence from the fluorescence sample generated by the irradiation of the excitation light, and the fluorescence-side concave diffraction grating 12 is provided on the fluorescence passage passing through the fluorescence-side incident slit 11. Deploy. As a result, the fluorescence is dispersed by the fluorescence-side concave diffraction grating 12, the course of the fluorescence is changed, and the fluorescence-side exit slit 13 is irradiated with the fluorescence. The fluorescence that has passed through the fluorescence-side exit slit 13 is received by the fluorescence-side photodetection element 14 arranged outside the slit. As the fluorescence side light detection element 14, for example, a photomultiplier tube or the like is used. Further, the output of the fluorescence side photodetection element 14 is connected to the preamplifier 15, and the output of the preamplifier 15 is the above A / D.
It is connected to the converter 8.

Then, the output of the A / D converter 8 is set to CP.
It is connected to U16 and the CPU 16 calculates the fluorescence intensity. That is, originally, the fluorescence intensity may be obtained based on the light intensity detected by the fluorescence-side photodetection element 14, but the cell 10 is affected by fluctuations in the light intensity itself emitted from the light source 1, noise in the optical system, or the like. The intensity of the excitation light with which the light is irradiated may fluctuate. Therefore, by using the light intensity of the excitation light detected by the excitation light-side photodetection element 14 as the reference light, the CPU 16 executes the following equation to obtain a pure fluorescence signal S without the influence of such fluctuations. ing. Then, the fluorescence signal S is
It is converted into analog through a D / A converter 17 connected to the CPU 16 and is output to a display device such as a CRT (not shown) or an output device such as various printers (not shown).

S = Em / Ex + c where Em: fluorescence intensity Ex: excitation light intensity (reference light intensity) c: coefficient for adjusting the baseline Here, in the present invention, a xenon-mercury lamp (Xe-Hg) is used as a light source. Lamp). This Xe-Hg lamp is formed by mixing and enclosing mercury and xenon gas in the lamp. Then, the same power consumption (15
The relationship between the emission intensity and the wavelength of the 0 W) xenon lamp and the Xe-Hg lamp was measured, and the result was as shown in FIG. The solid line in the figure shows the characteristics of the Xe-Hg lamp,
The broken line shows the characteristics of the xenon lamp. As is clear from the figure, Xg- in the ultraviolet region (400 nm or less)
The light intensity of the Hg lamp is much stronger (several times to several tens of times) than that of the xenon lamp. Further, since the light intensity in the visible region is almost the same, the same measurement as the conventional one can be performed. Further, since the calorific value is almost the same as that of the xenon lamp if the power consumption is the same, the heat dissipating mechanism is not particularly provided or the conventional one can be used as it is.

Since the light intensity in the ultraviolet region emitted from the light source is high as described above, the light intensity of the excitation light with which the fluorescent sample in the cell 7 is irradiated is also increased, and the fluorescence emitted from the fluorescent sample is accordingly increased. The light intensity of is also improved. And S
Since / N is generally proportional to the square root of the light source intensity, S / N is also improved in this example where the light intensity is strong. Also in this respect, high-sensitivity fluorescence measurement becomes possible.

Next, an experiment for confirming the effect of the present invention was conducted based on the above-mentioned embodiment. That is, a 150 W Xe-Hg lamp was used as a light source, the excitation wavelength was 365 nm, the fluorescence wavelength was 520 nm, and vitamin B1 was used.
Liquid chromatographic data and baseline noise data for (fluorescent sample) were obtained. The results are shown in FIGS. 3 (A) and 3 (B). On the other hand, as comparative data, a xenon lamp which has been conventionally used as a light source is used, and other configurations are measured for the same as above, and the results are shown in FIGS. 4 (A) and 4 (B).

The baseline noise is vitamin B.
The sensitivity was 32 times higher than that of the liquid chromatographic data data of No. 1. In addition, liquid chromatograph data,
It is a value based on the fluorescence signal S obtained by the CPU 16 described above, and indicates the relative intensity.

As is apparent from the figure, when the ratio of the baseline noise to the peak value, that is, the S / N is calculated, the S / N of FIG. 3 is about four times larger than the S / N of FIG. .

The specific structure of the above-mentioned fluorescence detecting device for liquid chromatograph is not limited to the above-mentioned embodiment, and various structures can be used. That is, the point is that the Xe-Hg lamp should be used as the light source.

[0017]

As described above, in the fluorescence detecting apparatus for liquid chromatograph according to the present invention, since the xenon-mercury lamp is used as the light source, the light intensity emitted from the light source in the ultraviolet region is high. Therefore, the light intensity of the excitation light with which the fluorescent sample in the cell is irradiated is also increased, and the light intensity of the fluorescence emitted from the fluorescent sample is also improved. Since the S / N is proportional to the square root of the light intensity, high sensitivity measurement is possible.

[Brief description of drawings]

FIG. 1 is a diagram showing a preferred embodiment of a fluorescence detection device for a liquid chromatograph according to the present invention.

FIG. 2 is a diagram showing characteristics of light intensity with respect to wavelengths of a Xe-Hg lamp and a xenon lamp.

FIG. 3 is a diagram showing experimental results for demonstrating the effect of the present invention, where (A) is liquid chromatograph data and (B) is baseline noise data.

FIG. 4 is a diagram showing experimental results for demonstrating the effect of the present invention, (A) is liquid chromatograph data, and (B) is baseline noise data.

[Explanation of symbols]

 1 Light Source 2 Condenser Lens (Optical System) 3 Excitation Side Entrance Slit (Optical System) 4 Excitation Light Side Concave Diffraction Grating (Optical System) 5 Beam Splitter (Optical System) 6 Excitation Side Photo Detector (Means to Detect Light Intensity ) 7 preamplifier 8 A / D converter (means for performing arithmetic processing) 9 exit slit (optical system) 10 cell 11 fluorescence-side entrance slit 12 fluorescence-side concave diffraction grating 13 fluorescence-side exit slit 14 fluorescence-side photodetector (light intensity Detecting means) 15 Preamplifier 16 CPU (means for performing arithmetic processing) 17 D / A converter

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Toshihiko Miyaji 5 2967 Ishikawa-cho, Hachioji-shi, Tokyo Inside JASCO Corporation

Claims (1)

[Claims] 1. A light source, a cell in which a fluorescent sample is placed, an optical system for irradiating the fluorescent sample in the cell with light emitted from the light source, a light intensity of the light before the irradiation, and the cell. In a fluorescence detection device for a liquid chromatograph, which is provided with a means for respectively detecting the light intensity of fluorescence emitted from the fluorescent sample inside, and means for performing a predetermined calculation process based on both light intensities detected by the detection means. A fluorescence detection device for a liquid chromatograph, wherein the light source comprises a xenon-mercury lamp in which xenon and mercury are mixed and sealed.