JPH05149878A - Laser fluorescence detector - Google Patents

Abstract

(57) [Summary] [Purpose] As a laser fluorescence detection device used for micro liquid chromatography or capillary electrophoresis, it can scatter scattered light more effectively than conventional devices.
An apparatus capable of detecting fluorescence under a high S / N ratio is provided. A laser light source that is linearly polarized in a predetermined direction is used as a light source for exciting a solution from a column, and a polarization filter that blocks polarized light in that direction is provided in front of a fluorescence detection sensor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a detection device based on a laser excitation fluorescence detection method, which is used in a micro liquid chromatograph or a capillary electrophoresis device as a separation analysis device.

[0002]

2. Description of the Related Art Micro liquid chromatography (MLC) using a capillary column with an inner diameter of 500 μm or less
Alternatively, in a laser fluorescence detection method used in a capillary electrophoresis apparatus (CE), conventionally, a laser beam is focused and irradiated onto a fused silica capillary from which a polyimide coating has been peeled off, and this laser light irradiation causes a capillary cell to be irradiated. In order to detect the fluorescence generated from the sample inside, Rayleigh scattered light or Raman scattered light is cut using an interference filter such as a bandpass filter, and only the desired fluorescence is selectively guided to, for example, a photomultiplier tube. The method has been adopted.

[0003]

By the way, in such a fluorescence detection method, scattered light is sufficiently cut,
An important point is to devise a method for detecting only the desired fluorescence, but it is desirable to cut scattered light as efficiently as possible by constructing an optical system by making good use of the characteristics of laser light.

The present invention has been made from this point of view, and can more effectively cut scattered light as compared with a conventional detection device, and detects fluorescence under a high S / N ratio. The purpose of the present invention is to provide a device that can be used.

[0005]

In order to achieve the above object, the laser fluorescence detection device of the present invention is an excitation light source that emits linearly polarized laser light as a laser light source for irradiating a solution from a column. And is provided in front of the optical sensor with a polarization filter that blocks light polarized in the above direction.

[0006]

When the capillary surface is irradiated with laser light linearly polarized in a predetermined direction, the reflected light from the capillary surface also has polarization. This reflected light that interferes with fluorescence detection can be effectively cut by a polarizing filter having a polarization plane different from the polarization direction of the reflected light, most preferably the direction perpendicular to the polarization direction of the reflected light. It is possible to prevent the light from entering the optical sensor for detecting fluorescence.

[0007]

EXAMPLE FIG. 1 is a schematic perspective view showing the configuration of an example of the present invention. The light (wavelength 442 nm, output 10 mW) from the helium-cadmium (He-Cd) laser 1 is
It is polarized in the vertical direction with respect to the optical axis, is guided to the focusing lens 5 via the mirror 2, the beam splitter 3, and the mirror 4, and is condensed on the capillary 6.

Incidentally, a part of the laser light extracted by the beam splitter 3 provided between the mirrors 2 and 4 is guided as a reference light to the photodiode 9 through the diffuser plate 7 and the iris diaphragm 8 and the light source. The light intensity of is monitored.

When the sample in the capillary 6 is excited by the irradiation of laser light and emits fluorescence, the fluorescence is collected by the condenser lens 10 placed in the direction orthogonal to the irradiation optical axis of the laser light. Collected, iris diaphragm 11, polarization filter 12 and bandpass filter 13
It is guided to the photomultiplier tube 14 via (490DF20),
Its intensity is detected.

Here, the polarization filter 12 is arranged in such a posture that its polarization plane is orthogonal to the polarization direction of the light from the laser 1. In the above configuration, the condensing lens 10 arranged close to the capillary 6 collects not only the fluorescence from the sample but also the laser light scattered from the wall of the capillary 6.

Of the scattered light from the wall of the capillary 6, the reflected light from the surface of the capillary on the incident side is
Since the polarized light is kept in the above-mentioned direction, the portion is cut by the polarizing filter 12 and does not reach the bandpass filter 13 and the photomultiplier tube 14.

In order to evaluate the performance of the examples of the present invention as described above, acetonitrile / phosphate buffer (10 mM, p
H7) = 70/30 is enclosed in a capillary 6, and a laser from a laser 1 is radiated to this to photomultiplier tube 1
When the output current of No. 4 was measured, the iris diaphragm 11 and the photomultiplier tube 1 without the polarization filter 12 from the configuration of FIG.
4 was 23.5 nA when only the bandpass filter 13 was placed between 4 and 2.3 nA when the polarization filter 12 was added as shown in FIG.
It was confirmed that the value was reduced to 0, and the background was significantly reduced. At the same time, the magnitude of noise is
By inserting 2, 0.35nA to 0.075nA
It fell to 1/4.

Next, 10 ^-10 M coumarin is sealed in the capillary 6 and irradiated with laser light, and the fluorescence from this coumarin is measured as a signal. When the bandpass filter 13 alone is 20 nA, the bandpass filter 13
6.5 nA when combined with the polarizing filter 12
Met.

Table 1 is a summary of the above evaluation results.
As is clear from this table, although the signal was reduced to ⅓, the total S / N ratio was improved to 1.5 times due to the reduction in background and the accompanying reduction in noise.

Here, the position of the polarization filter 12 is not limited to the position shown in FIG. 1, and it may be provided between the capillary 6 and the photomultiplier tube 14. However, an interference filter such as a bandpass filter, In general, since it emits a very small amount of fluorescence, it is preferable to make the incident light intensity on such a filter as small as possible. Therefore, as shown in FIG. 1, the deflection filter 12 should be arranged in front of the bandpass filter 13. Is desirable.

The light emitted from the laser light source is sometimes randomly polarized light, and its polarization plane may change with time. In this case, for example, by arranging a polarizing prism at the exit of the laser beam, linearly polarized light,
Moreover, a laser beam with a constant plane of polarization can be obtained. As described above, by using the polarizing prism, the present invention can apply almost all lasers as the laser used for the light source.

[0017]

As described above, according to the present invention, the laser light for irradiating the capillary is linearly polarized in a predetermined direction, and the front stage of the optical sensor for detecting the fluorescence generated from the sample in the capillary. In addition, since a polarizing filter that cuts the polarized light in the above direction is provided, it becomes possible to effectively block the laser scattered light from the capillary in front of the optical sensor, which significantly reduces the background value and noise. Can be reduced, and the S / N ratio can be improved.

[Brief description of drawings]

FIG. 1 is a schematic perspective view showing the configuration of an embodiment of the present invention.

[Explanation of symbols]

 1 ... He-Cd laser 5 ... Focusing lens 6 ... Capillary 10 ... Condensing lens 12 ... Polarization filter 13 ... Bandpass filter 14 ... ..Photomultiplier tubes

Claims (1)

[Claims] 1. In a micro liquid chromatography or a capillary electrophoresis analyzer, a solution emitted from a column is irradiated with laser light to excite a component contained in the solution, and fluorescence generated by the solution is applied to an optical sensor. A device for guiding and detecting, comprising: an excitation light source that emits laser light linearly polarized in a predetermined direction; and a polarization filter that is provided in front of the optical sensor and blocks light that is polarized in the direction. And a laser fluorescence detector.