JPH02227637A - Fluorophotometer - Google Patents

Abstract

PURPOSE:To exactly measure fluorescence with high accuracy by providing a concave mirror around an optical cell which is transparent to UV and visible light, passing a sample to be measured in the direction of the central line of the optical cell and passing excitation light along the central line. CONSTITUTION:A cell 3 made of quartz, an elliptical mirror 4 and an optical system 5 are provided in a casing 2. The eluate from the column of high- performance liquid chromatograph is admitted from a stainless steel pipe 11 into the cell 3 and is discharged from a stainless steel pipe 16. The eluate is excited by the incident excitation laser light from an optical fiber 6 to emit the fluorescence 2 while flowing in the cell 3. This fluorescence 25 is reflected by a reflecting surface 26 of the mirror 4 and is condensed to a focus 27. The fluorescence 25 collected at the focus 27 is collimated by a collimator lens 17a and the unnecessary scattered light is removed therefrom by a filter 18. This light is condensed by a focusing lens 17b onto a photoelectron multiplier 19. The current proportional to the condensed fluorescence obtd. by the multiplier 19 is amplified by an amplifier 21. The fluorescent intensity is determined and is recorded in a recorder 22.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a fluorometer, and more particularly to a fluorometer for a fluorescence analyzer. The present invention also relates to a fluorescence detector using a laser-excited fluorescence method for a liquid chromatograph analyzer, and more particularly to a fluorescence detector using a laser-excited fluorescence method for a high-performance liquid chromatograph analyzer.

(b) Conventional technology For example, in high performance liquid chromatography analyzers,
Because the fluorescence detection method is highly sensitive, it is possible to react not only fluorescent substances but also non-fluorescent substances with a fluorescent reagent and measure them as fluorescent reaction products with high sensitivity, so-called derivatization. Chromatography is being performed.

In this case, in fluorescence analysis using a laser-induced fluorescence method that uses a laser as a light source, the laser is generally applied perpendicular to the direction of flow of the eluent through an optical cell that is transparent to ultraviolet and visible light. The fluorescence emitted in the direction perpendicular to the direction of flow and the direction of the laser is measured (for example, Spectroscopy Research, Vol. 37, No. 5, 1988, No. 32).
), a method has also been proposed in which a laser is passed along the flow direction of the eluent flowing through an optical cell and the fluorescence emitted in a direction perpendicular to the flow direction of the eluent and the direction of the laser is measured. There is.

(c) Problems to be solved by the invention By the way, in the method of applying a laser along the direction of the flow of the eluent, the fluorescence emitted linearly and evenly in 360 degrees around the direction of the flow of the eluent is equivalent to the measurement. This method is superior to the method of applying a laser perpendicular to the direction of flow of the eluent, as it can increase the amount of fluorescence detected and measure smaller amounts of sample. Due to the optical arrangement in which a portion of the fluorescence is collected by a lens, passed through an optical filter, and then measured by a photomultiplier tube, the effective utilization rate of fluorescence is low, and its advantages are not fully utilized, which is a problem. ing.

An object of the present invention is to solve problems related to the effective utilization rate of fluorescence in a fluorometer that applies excitation light in the flow direction of a liquid to be measured such as an eluent.

(d) Means for Solving the Problems The present invention aims to provide a fluorometer that can measure the fluorescence emitted by applying excitation light in the flow direction of a liquid to be measured with a high effective utilization rate. .

The present invention is made of a transparent body for ultraviolet and visible light,
A cylindrical optical cell that has a transmitting surface at one end, the other end is shielded from light by contacting an opaque body, and an inlet and an outlet for the sample to be measured are provided at different positions in the direction of the center line of both end faces. , has an excitation light source located on the transmission surface side on the center line of both end faces of the optical cell, and has an axis of symmetry, the axis of symmetry is located in the same direction as the center line, and the concave portion is located in the same direction as the center line. A concave mirror located on the light source side and having a mirror surface surrounding the optical cell, and a photodetector located on the axis behind the opaque body with respect to the optical cell. The fluorometer is characterized by:

In the present invention, the optical cell is provided with an inlet and an outlet for the sample to be measured along the direction of the center line so that the sample to be measured flows along the center line of both end faces of the optical cell. 6 In the present invention, the optical cell has an end face on the incident side of the excitation light such as a laser that is transparent to the excitation light, but the other end face is formed so that the incident excitation light does not pass through. , covered by an opaque body.

In the present invention, the optical cell emits not only fluorescence but also Raman scattered light due to the solvent, so when measuring a trace amount of fluorescence, the filter installed in the photodetector is The area around the cell is shielded from light with light-shielding paint, etc., except for the exit of the fluorescent light.

In the present invention, 1. so that the fluorescence emitted from the optical cell can be effectively used for measurement. The optical cell is surrounded by a concave mirror 6 In the present invention, the concave mirror reflects the fluorescence emitted from the optical cell and focuses it on the photodetector within the concave mirror, and the concave mirror is symmetrical about the axis of symmetry. For example, there are ellipsoidal mirrors, parabolic mirrors, spherical mirrors, etc., which have a spherical or aspherical reflecting surface formed in .

Among these concave mirrors, the ellipsoidal mirror is such that all the light emitted from one focal point is concentrated at the other focal point, so the fluorescence exit of the optical cell is located at one focal point of the ellipsoidal mirror, and outside the other focal point. By providing a photodetector in the optical cell, it is possible to simply and easily perform photometry of all the fluorescence emitted from the periphery of the optical cell, which is preferable for use in the present invention.

In the present invention, the light source may be a tungsten lamp, a xenon arc lamp, a mercury lamp, or a mercury lamp, as in conventional fluorescence analysis.
Mercury-cadmium lamps, Zn-Cd-H lamps, laser lights, etc. are used.

In the present invention, a photomultiplier tube, a phototransistor, and its narrow light sensor can be used as the photodetector.

(E) Function The present invention provides a concave mirror around an optical cell that is transparent to ultraviolet and visible light, allows the sample to be measured to flow in the direction of the center line of the optical cell, and allows excitation light to pass along the center line. Therefore, the fluorescence emitted uniformly around the optical cell by the sample to be measured is reflected by the concave mirror and collected, and can be easily measured by a photodetector.

Moreover, in the present invention, since the exit side of the excitation light of the optical cell is shielded from light by an opaque material, the influence of scattering of the excitation light on the photodetector can be reduced, and the photometric measurement of fluorescence for a minute amount of sample is possible. , can be performed accurately, with high precision, and easily.

(F) EXAMPLES Hereinafter, examples of embodiments of the present invention will be described with reference to the accompanying drawings, but the present invention is not limited in any way by the following explanations and exemplifications.

FIG. 1 is a schematic explanatory diagram of an embodiment of the present invention, shown partially broken away.

In FIG. 1, a fluorometer 1 includes a casing 2, a quartz cell 3, an ellipsoidal mirror 4, and an optical system 5.
In this example, a laser beam is used as a light source for excitation light, and the laser beam is guided through an optical fiber 6 from a laser source (not shown). The optical fiber 6 is fixed in a liquid-tight or air-tight manner to a fiber joint 8 attached to the concave side 7 of the ellipsoidal mirror 4 by means of a member 9, for example, by screwing. The fiber joint 8 has a member 10 at which the cell is attached at a position opposite to the position at which the optical fiber is attached.
The mounting is fixed in a liquid-tight or air-tight manner, for example, by screwing.

The tip of the optical fiber 6 is in contact with the cell 3 that is attached to the fiber joint 8 in a liquid-tight or air-tight manner, and the cell 3
and optical fiber 6 are coaxially connected. Therefore, the laser beam from the optical fiber 6 can easily enter the cell 3 and act on the sample within the cell 3 as excitation light.

A stainless steel tube 11 connected to a column (not shown) of a high performance liquid chromatograph is fixedly attached to the fiber joint 8 by a stainless steel tube attachment member 12. In this stainless steel pipe attachment, the member 12 is connected to a connecting member 14 surrounding the tip 13 of the cell 3, and this connecting member 14 connects the optical fiber 6 to the stainless steel pipe 1.
1 and cell 3 are connected in a liquid-tight manner.

The other end of the cell 3 is connected to a stainless steel pipe 16 by a stainless steel pipe connecting member 15.

For convenience of explanation, behind the stainless steel pipe connecting member 15,
An optical system 5 composed of a collimator lens t7m, a converging lens 17b, and a filter 18 such as a band pass filter or a sharp cut filter is provided, and a photomultiplier tube 19 is provided behind the optical system 5.

The output section 20 of the photomultiplier tube 19 is connected to an amplifier 21, and the amplifier 21 is connected to a recorder 22. Further, the input section 23 of the photomultiplier tube 19 is connected to a high voltage power source 24.

Since the present example is constructed as described above, the eluent from the column of the high performance liquid chromatograph is caused to flow into the cell 3 through the stainless steel tube 11 connected to the column, and is discharged from the stainless steel tube 16. While the eluent flows through cell 3,
It is excited by the excitation laser light incident from the optical fiber 6 and emits fluorescence 25. This emitted fluorescence 25
is reflected by the reflecting surface 26 of the ellipsoidal mirror 4 and condensed at the focal point 2.

The fluorescence 25 collected at the focal point 27 is collimated by the first collimator lens 1a, unnecessary scattered light is removed by the filter 18, and is focused onto the photomultiplier tube 19 by the next converging lens 17b. Ru. In the photomultiplier tube 19, a current proportional to the focused fluorescence is obtained. This current is then amplified by an amplifier 21, and the fluorescence intensity is determined and recorded on a recorder 22.

In this example, all the fluorescence emitted in an angular region of 360 degrees around the central axis of the cell 3 is focused by the ellipsoidal mirror 4 at the focal point 27 and measured by the photomultiplier tube 19. The amount of fluorescence measured can be increased.

In this example, the cell 3 is not provided with a mask, but the fluorescent light 25 linearly emitted from the cell 3 is reflected at the focal point 2 on the concave side 7.
8 is focused near the other focal point 2, but as the distance away from the focal point 28 on the concave side 7 increases,
Since there is a possibility that the light will not be focused near the other focal point 27 and become stray light, it is preferable to form a mask in the area where the distance from the four-side focal point 28 of the cell 3 is large. Formation of this mask is preferable because it prevents the resolution of the high performance liquid chromatograph from deteriorating due to the measurement of excessively long linear fluorescence.

In this example, laser light from a laser light source is guided through an optical fiber and used as excitation light, but an optical system including a lens or a reflecting mirror may also be used to guide the excitation light. In addition, the light source of the excitation light is not a laser (incoherent light such as a Xe lamp can also be used.Also, in this example, a cell made of quartz is used as a cell, but optical glass such as Pyrex glass etc. A material that is transparent to excitation light and fluorescence and corrosion resistant to non-measured liquids can be used.

(g) Effects of the invention The present invention provides a concave mirror around an optical cell that is transparent to ultraviolet and visible light, allows the sample to be measured to flow in the direction of the center line of the optical cell, and emits excitation light along the center line. Because it passes through the fluorescent light, fluorescence can be used more effectively than with conventional fluorometers, and the amount of fluorescence that can be measured can be dramatically increased.

Therefore, according to the present invention, it is possible to measure even trace amounts of samples for which it is difficult to measure fluorescence using conventional fluorometers.
Accurate and highly accurate fluorescence measurement becomes possible, and the analysis accuracy and analytical range of fluorescence analysis can be expanded.

[Brief explanation of the drawing]

FIG. 1 is a schematic explanatory diagram of an embodiment of the present invention, shown partially broken away. The symbols in the figure are 1: fluorometer, 2: casing, 3: quartz cell, 4: ellipsoidal mirror, 5: optical system, 6: optical fiber, 7: concave side, 8: fiber joint, 9: Fiber attachment is a member, 10 is a cell attachment is a member, 11
1 is a stainless steel pipe, 12 is a stainless steel pipe installation member, 1
3 is the tip of the cell, 14 is a connecting member, 15 is a stainless steel tube connecting member, 1B is a stainless steel tube, 1a is a collimator lens, 17b is a converging lens, 18 is a filter, 1
9 is a photomultiplier tube, 20 is an output part of the photomultiplier tube, 21
22 is an amplifier, 22 is a recorder, 23 is an input part of a photomultiplier tube, 24 is a high voltage power supply, 25 is fluorescence, 2B is a reflecting surface of an ellipsoidal mirror, and 27 and 28 are focal points.

Claims (1)

[Claims] It is made of a material that is transparent to ultraviolet and visible light, has a transmitting surface at one end, and is shielded from light by contacting an opaque material at the other end, and has an inlet and an outlet for the sample to be measured at different positions in the direction of the center line of both end surfaces. A cylindrical optical cell is provided with a cylindrical optical cell, and an excitation light source is located on the transmission surface side on the center line of both end faces of the optical cell, and has an axis of symmetry, with the axis of symmetry being centered at the center. a concave mirror located in the same direction as the line, with its concave portion facing the light source side and having a mirror surface surrounding the optical cell; and a concave mirror located on the axis behind the opaque body with respect to the optical cell. A fluorometer characterized by comprising a photodetector provided with a photodetector.