JP3136574B2 - Spectral characteristics measuring device for trace liquid samples - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for measuring a very small amount of a sample liquid in a spectrophotometer for measuring a spectral characteristic of a sample in a visible / ultraviolet region or the like.

[0002]

2. Description of the Related Art A visible / ultraviolet spectrophotometer is an analytical instrument widely used for various applications in the field of analytical chemistry. Although its application field is wide-ranging, its main purpose is to measure the spectral transmission characteristics of a sample, which is generally a liquid, in units of absorbance that is proportional to the concentration of the sample, and to analyze the sample qualitatively or quantitatively. Often do. With the progress of so-called biotechnology-related technology in recent years, the role played by a spectrophotometer in the field of biochemical analysis has increased as in other conventional fields. Therefore, as an issue necessary for spectroscopic analysis in the biochemical field including analysis of proteins, DNA, and the like, the number of samples to be measured is extremely small and valuable and expensive. It has come to be said that qualitative or quantitative analysis must be performed. In particular, in DNA-related analysis, the total sample volume is often several hundred μl or less, and further, the sample is used for measurement by a spectrophotometer in order to disperse the sample for use in various analyzers and the next-stage research. The volume ranges from a few μl to at most
In most cases, it is less than or equal to 1. Conventionally, as means for measuring the absorbance of such a small amount of liquid, a spectrophotometer in which the luminous flux size in measurement analysis is made as small as possible by a luminous flux stop or a condensing optical system, or an accessory device therefor And a special sample container for supporting a micro liquid sample on the measuring section.

[0003] Several types of special sample containers that have been used in the past are known. Among them, the most widely used one is the optical path length used in ordinary spectroscopic analysis. Based on the shape of a 10 mm square cell, a method using a special square cell with a reduced optical path width and optical path height to reduce its internal volume, and introducing a sample by capillary action into a thin tube made of quartz glass, There has been known a method in which a central axis is perpendicular to the measurement optical axis of the spectrophotometer and supported by a measurement unit of the spectrophotometer, and a condensing means such as a cylindrical lens is used in combination. As means for supporting and fixing these containers to the sample measuring portion of the spectrophotometer, a commonly used 10 mm square cell holder is basically used, and an improved version thereof or a method for positioning and fixing a thin tube by a leaf spring or the like is used. Etc. were known.

[0004] Each of these conventional techniques has the following problems. First, a description will be given of a minute amount cell of a square cell which is a first conventional example. FIG. 8 shows a general schematic structure of a conventional square minute amount cell. This square micro-quantity cell 21 has a bottom surface (a × b in FIG. 8A) of the same shape as a normal 10 mm square cell for spectroscopic analysis, and has a structure in which the volume of a portion for inserting a sample is as small as possible. It is. The reason why the bottom surface shape is made the same as that of a normal square cell is that this cell can be held by a normal square cell cell holder. In the example of FIG. 8, the portion where the sample is placed, that is, the measurement unit 22 has dimensions = height h × optical path width w.
(See FIG. 8B) and the portion of the optical path length d (see FIG. 8A). The tapered portion 23 formed on the upper part of this portion is provided for facilitating the introduction and discharge of the sample using a pipettor, a syringe, or the like. FIG. 8B
Shows a GG section in FIG. 8A, and FIG. 8C shows a state in which the sample liquid 3 is injected into FIG. 8B. Conventionally, when a small amount of liquid is measured using the square minute amount cell 21, a normal square cell provided in the measuring unit of the spectrophotometer is held after a sample is injected into the measuring unit 22 by a pipettor or the like. The cell 21 for the square minute amount is fixed to the cell holder to be measured, and the measuring light K is passed through the light beam stop 5 having an appropriate shape.
The measurement was performed after limiting the luminous flux to be applied only to the measurement unit 22. In this case, the size of the luminous flux restricted by the luminous flux stop 5 is different from other parts of the spectrophotometer, for example,
Since it is necessary to satisfy the optical performance required for a light source spectrometer, a photometric system, a detector, and the like, the size cannot be reduced beyond a certain limit, and the limit is 1 mm × in a normal spectrophotometer.
It is about 1 mm or φ1 mm. Therefore, the sample is illuminated with a light beam of this size. Further, in consideration of the position reproduction accuracy due to the attachment / detachment of the square minute amount cell 21, the dimension of the measuring unit 22 of the square minute amount cell 21 is approximately w =
The lower limit was about 2 mm, h = 2 to 2.5 mm.
Further, when the sample liquid 3 is injected into the square minute amount cell 21, the liquid surface does not become a flat surface due to surface tension, but generally forms a concave surface as shown in FIG. Required more sample volume than was needed.

[0005] From these optical restrictions and restrictions due to the liquid surface shape, if the optical path length d of the cell is d = 10 mm,
The minimum required liquid volume for measurement was about 50 μl as a lower limit, and it was extremely difficult to measure with a liquid volume below this. In addition, although the tapered portion 23 is formed in the rectangular microvolume cell 21 so as to be convenient for injecting the sample liquid, the space into which the liquid is injected is small when the sample liquid 3 once injected or the cell is washed. Therefore, it was extremely difficult. Furthermore, to enable measurement in the ultraviolet region required in the field of biochemistry,
The cell is generally a fusion-bonded product of quartz glass, but has the disadvantage that the price of the cell is extremely high because the material is expensive and difficult to process.
As a method of further reducing the required liquid amount by applying this cell, a method of reducing the optical path length d can be considered. For example, in order to reduce the sample amount to 5 μl, d must be set to 1 mm. Since the area of the liquid inlet is greatly reduced, the injection and collection of the sample and the washing of the cell are performed using the above d =
This became even more difficult than in the case of 10 mm, and was not a practical method. In addition, in order to save the labor of collecting the sample and washing the cell, a method of disposing the cell may be considered, but if the cell itself is extremely expensive as described above and the sample cannot be collected, the method may be used. Since it is required that the liquid volume be reduced to several μl or less, this was also practically impossible.

FIG. 10 shows a method using a quartz tube which is a second conventional example. In this method, a sample liquid 3 is sucked into a capillary tube 31 made of quartz glass by utilizing a capillary phenomenon.
This is a method in which a tube axis is installed in a measuring section of a spectrophotometer so as to be orthogonal to the measuring optical axis, and a condensing optical system such as a cylindrical lens 32 shown in FIG. According to this method, the introduction of the sample liquid, which is a problem in the rectangular cell, can be relatively easily performed by utilizing the capillary phenomenon. Further, the collection of the sample liquid from the quartz tube and the washing of the tube can be easily performed by using air pressure or the like. However,
In the case of this method, since the quartz tube 31 having a generally cylindrical shape and the sample liquid 3 held therein function as a kind of cylindrical lens, the shape of the light beam entering and exiting the tube 31 is determined by another part of the spectrophotometer. However, there is a problem that a cylindrical lens 32 or an optical system equivalent thereto is necessarily required for shaping according to the above. For example, in order to set the sample volume to 5 μl and the liquid level of the sample to 3 mm, the inner diameter of the quartz tube must be about 10.4 mm, and an extremely strong cylindrical lens with a radius of curvature of about 0.7 mm is measured. A problem arises in that an optical system for correcting this need to be added as being installed in the optical path. In addition, the quartz tube 31 requires expensive quartz glass having a transmittance even in the ultraviolet region in order to provide optical performance enough to withstand spectral transmission measurement on both the inner and outer surfaces, that is, surface roughness, shape accuracy, and the like. Material costs,
There was a problem that the processing cost would be expensive. Furthermore,
Since the outer wall of the thin tube has a large curvature, it is extremely difficult to prevent the measurement light from passing through the tube wall of quartz glass. Therefore, there has been a problem that the accuracy of the absorbance measurement is reduced.

[0007] The conventional means for fixing these containers to the measuring part of the spectrophotometer also has the following problems. FIG. 9 shows a conventional example of a cell holder for a rectangular micro cell.
As in the case of FIG. 8, the square minute amount cell 21 into which the sample liquid 3 has been injected is inserted into the cell holder 24 and fixed by the leaf spring 17. The cell holder 25 is fixed to a base plate 26, and is fixed to a sample measuring section of the spectrophotometer by a mounting hole 26A. Reference numeral 27 denotes a cell height adjusting mechanism using a screw or the like, which adjusts in the direction of arrow E in FIG. 9 to change the cell insertion depth. As described above, this mechanism has a very small sample amount, and the liquid to be measured is often slightly larger than the light beam stop 5 through which the light beam passes. At a position in the height direction where the luminous flux is illuminated,
This is for adjusting the square minute amount cell 21. Since the cell 21 is very small, if the positioning leaf spring 25 is made too strong,
1 is difficult to attach and detach, and if the leaf spring 25 is weakened, there is a problem that the position reproducibility when the cell 21 is attached and detached deteriorates. In addition, the height must be adjusted by the adjusting mechanism 27 in accordance with the amount of the sample liquid injected, and furthermore, if there is play in the adjusting mechanism portion, it is also said that the reproducibility when the cell 21 is detached is also poor. There was a problem. Furthermore, since a small cell is accurately positioned and fixed, and an adjusting mechanism is required, the entire configuration is complicated and the cost is high.

FIG. 11 shows a second conventional example of a quartz tube holder. As shown in FIG. 11, the thin tube 31 into which the sample liquid 3 has been sucked is inserted in accordance with the V groove 33A of the holder 33, and is fixed by the presser spring 24 attached to the holder 33. The attachment to the spectrophotometer is performed through the attachment hole 26A of the base plate 26 as in FIG. Reference numeral 32 denotes a cylindrical lens for correcting a lens effect by the same thin tube as shown in FIG. This holder 33
According to the above, it is possible to facilitate the attachment and detachment by making the length of the thin tube longer than that of the measuring section, and the positioning mechanism using the V-groove 33A accurately positions the thin tube 31 on the measuring optical axis. can do. Since the introduction of the sample liquid 3 into the thin tube is easier than in the case of the rectangular microcell 21, the liquid level can have a margin as compared with the light beam stop 35, and a height adjusting mechanism can be provided. It can be unnecessary. Although this method has the above advantages, the structure has the following disadvantages. Thin tube 3
1 is a thin-walled small-diameter quartz tube, which is difficult to handle and may be damaged during desorption.
In order to correct the lens effect of the thin tube, a cylindrical lens 32 which is an expensive aspherical element is required, and a V-groove portion and a pressing spring mechanism for accurately positioning and fixing the thin tube 31 in an axial direction having a relatively large dimension are provided. In some cases, high-precision processing is required.

[0009]

SUMMARY OF THE INVENTION The present invention relates to a spectrophotometer for measuring the spectral characteristics of a sample in the visible / ultraviolet region, which is used as a cell material when the amount of the liquid sample to be measured is extremely small. It is an object of the present invention to be able to measure its spectral characteristics without using expensive components and without requiring a special auxiliary optical system.

[0010]

The liquid to be measured is sucked and held from one end of the thin tube by capillary action or suction means. In this state, the suction side of the thin tube includes a portion holding the liquid to be measured. Cut the thin tube at an appropriate length from the end, and fix the cut part of the thin tube holding the liquid to be measured to the measuring section of the spectrophotometer so that the center axis is parallel to the measuring optical axis. The spectral characteristics of a trace liquid sample were measured.

[0011]

According to the present invention, the sample solution is injected into the capillary by sucking the sample solution by capillary action or a suction means, and the capillary portion filled with the sample solution is cut off. The tubule section is placed in the measuring section such that the central axis is parallel to the measuring optical axis. Therefore, the diameter of the sample liquid can be determined by the inner diameter of the thin tube, and the thin tube can be made thinner in any number of ways, but as described above, the luminous flux size in the measuring section of the spectrophotometer should be further reduced. There is a limit that is extremely difficult in practice, and this limit is temporarily set to φ1 mm. Considering some margin from the position accuracy and position reproducibility when the thin tube fragment 4 is supported by the spectrophotometer measurement unit, the inner diameter of the thin tube 1 is set to φ2.
mm, and the optical path length d = 1 mm, the amount of the sample liquid 3 held in the capillary tube fragment is about 3 μl.
If the inner diameter is about 2 mm, it depends on the properties of the sample.
Suction up to about m is sufficiently possible. In the case of the present invention, the measurement light is transmitted only by the sample liquid if the luminous flux size is sufficiently limited and the diameter of the thin tube is appropriately selected, and there is no cell wall before and after the sample liquid, and therefore, Even if the measurement in the ultraviolet region is required, the thin tube does not need any optical characteristics such as transmission characteristics and surface roughness, and in extreme cases, it can be made of opaque material Therefore, various inexpensive materials and manufacturing methods can be appropriately selected and manufactured. Specifically, a glass molded product or an injection or extruded product of a hard plastic such as styrene or acrylic is suitable. However, in some cases, since the thin tube does not require optical characteristics, it is possible to use metal such as stainless steel or ceramic. Also, it is easy to prevent the measurement light beam from passing through the cell wall around the sample, so that only the light transmitted through the sample can be measured purely. The required sample volume is extremely small as described above and the capillary 1
Can be manufactured at low cost, and if it is not necessary to collect the sample, it is possible to discard the entire thin tube fragment 4. Also, if collection is necessary,
The sample liquid 3 may be collected from the thin tube fragment 4 by air pressure or the like, and only the thin tube fragment 4 may be discarded, so that the trouble of cleaning the container which has been a problem with the conventional square minute amount cell or the like is unnecessary. Also in this case, the remaining portion of the thin tube 1 other than the fragment 4 can be used repeatedly as described above as long as its length is practical.

[0012]

FIG. 1 shows an embodiment of the present invention. First, as shown in FIG. 1A, one end of a thin tube 1 is slightly immersed in a liquid surface of a sample container 2 containing a sample solution 3 synthesized by a reaction device or the like, and the sample solution 3 is made utilizing a capillary phenomenon.
Is introduced into the thin tube 1. At this time, vacuum pump, dropper,
A suction means such as a syringe may be used in combination. As shown in FIG. 2, the rear end of the thin tube 1 is thickened, and a rubber ball 8 is attached.
The thin tube itself may be like a Komagome pipette. The sample solution does not need to be filled over the entire thin tube, and depending on the absorbance of the sample solution, it is sufficient if the sample solution is sucked to a height of about 0.5 to 1 mm. Even if the thin tube 1 is separated from the liquid surface in this state, the sample liquid 3 is held in the thin tube 1 by surface tension. Next, as shown in FIG. 1B, the thin tube 1 is cut at a fixed length h from the end face. The length h may be determined as appropriate depending on the state of liquid filling, and may be determined, for example, at a position intermediate between the end face of the thin tube and the liquid level in the thin tube. As a method of cutting the thin tube 1 at a predetermined length,
As shown in FIG. 2, an engraved line 6 is provided on the outer circumference of the thin tube 1 at a predetermined interval d (equal to h in FIG. 1B), and the thin tube fragment 4 for measurement can be easily cut off with the engraved line 6. The interval d between the engraved lines may be selected so as to have a predetermined measurement optical path length in accordance with the characteristics of the liquid, the diameter of the tube, and the like. The material and the manufacturing method are not optically restricted, and accordingly, the thin tube is made of a material having a suitably low fracture brittleness, that is, a material in which the thin tube piece 4 can be easily broken, for example, glass, hard plastic, or the like. It becomes easy to cut off at the one marking line 6. Further, the remaining portion of the thin tube can be used for the next measurement as long as the entire length L of the thin tube can sufficiently be used. The inner diameter D of the thin tube may be appropriately selected and determined in advance according to the properties of the sample liquid and the amount of the measurement liquid.

FIG. 3 shows an example of a jig for setting the above-described sample cell of the present invention at a predetermined position. This jig has a square shape with a bottom surface of 10 mm × 10 mm and is adapted to a cell holder for setting a normal cell of a spectrophotometer. A V-groove 7 is formed on the upper surface, and when the above-described sample cell is placed on the V-groove, the axis of the cell 4 automatically coincides with the optical axis of the measurement light of the spectrophotometer.

FIG. 4 shows an example of a thin tube fixing device for fixing a thin tube to a jig at the time of measurement, which also serves as a tool for breaking off a portion where a sample is sucked from a thin tube with a score line. The sample liquid 3 is sucked into the thin tube 1 as shown in FIG. 2 by the method as shown in FIG. 1A or FIG.
The V-groove 11A, which is placed so as to match the one V-groove 11A, has a length substantially equal to the scribe line interval d of the thin tube, and a space 11B for cutting is formed behind the V-groove 11A. Fixing member 12
On the lower member 11. Upper member 12 and lower member 11
A V-groove portion 12A is formed in the same manner as
As in 1A, it is substantially equal to d, and a breaking space 12B is formed in the rear similarly to the lower member 11. The thin tube 1 is positioned by being sandwiched between the two V-grooves 11A and 12A. The upper and lower members 11 and 12 are fixed by fixing screws 13. A breaker 14 has a through hole 14A at the center thereof, and the hole diameter is set slightly larger than the outer diameter of the thin tube 1. The cutting tool 1 is previously attached to the thin tube 1 fixed by the fixing tool upper and lower members 11 and 12.
4 is inserted. A thin tube 1 is inserted into the V-grooves 11A and 12A.
FIG. 5 shows a cross-sectional view of the center of the thin tube shaft in a state where is inserted.

In FIG. 5, the folding tool 14 holds the thin tube 1 therein and is inserted into the folding spaces 11B and 12B formed by the fixing tool upper and lower members 11 and 12. The thin tube 1 has a fixing member 11,
12 fixed. In this state, the breaker 14
5 in the direction of the arrow in FIG. 5, the thin tube 1 is broken off by a predetermined dimension d and left in the fixture upper and lower members 11 and 12,
Other parts can be removed. Reference numeral 3 denotes a sample liquid sucked into the thin tube 1. According to this fixing method, since a short section of an axisymmetric cylindrical tube may be fixed so that its axis is at a predetermined position, when fixing a rectangular cell having a rectangular parallelepiped shape, or when a small length of a thin tube is used. Compared to a conventional example in which a certain fragment is positioned so that its axis is perpendicular to the optical axis, highly accurate positioning and fixing can be achieved with a simpler structure. FIG. 1C shows the tubule fragment 4 filled with the liquid thus cut. As shown in FIG. 1D, the thin tube section 4 is supported by a measuring section of the spectrophotometer so that its central axis is parallel to the measuring optical axis, and a light beam stop 5 for restricting a light beam size is used as needed. Then, the absorbance of the sample solution 3 is measured.

FIG. 6 shows an example of the fixing member on the spectrophotometer side in this case. The broken and fixed thin tube fragment 4 is attached to the measuring section of the spectrophotometer together with the fixing member upper and lower members 11 and 12. A fixture holder 15 is attached to the spectrophotometer measurement unit via an attachment hole 15A, and a fixture combination product A holding the thin-tube fragment 4 is held by a leaf spring 16 of the holder 15.
Is positioned and fixed. Reference numeral 17 denotes a light beam stop. In this example, the fixture combination A is fixed in a similar manner to the case of the conventional square cell, but unlike the case of the square cell, the fixture combination A is restricted in shape and material. Since there is no such arrangement, for example, the positioning accuracy and the reproducibility of attachment and detachment can be easily improved as compared with the case of a square cell holder by forming a positioning pin or a positioning groove, or fixing with a fastening component such as a screw.

FIG. 7 shows a fixture and a fixture holder when positioning pins and screws are used. Fixture combination product A '
Has a positioning hole 18 formed therein, and is fitted to a positioning pin 20 fixed on a base plate 19 attached to a spectrophotometer (not shown) to position the fixture combination product A ′. I do. Further, in order to secure the fixing, the fixing device combination product is fixed with the fixing screw 13 '. According to this method, the positioning and fixing of the fixture can be performed accurately and easily. The remaining capillary remaining by the method shown in FIG. 5 is collected or discarded by blowing out the sample liquid remaining at the tip, or by further cutting and discarding the portion including the liquid residue to obtain the remaining liquid. The portion can be used repeatedly while its length withstands use.

FIG. 1E is a diagram showing a cross section of the thin tube fragment 4 in a measurement state. The sample solution 3 in the thin tube fragment 4 forms a curved surface due to surface tension. However, if the diameter of the thin tube 1 is appropriately selected, the sample solution 3 does not have an extremely curved surface which is problematic in the square microcell shown in the above conventional example. Since the liquid surface has a curvature, the effect of the sample liquid 3 acting as a lens can be reduced to such an extent that there is no problem in measurement. Even if an optical system for correcting the lens effect due to the curved surface formed by the liquid surface is added, the curved surface is axially symmetric with respect to the optical axis, so that the correcting optical system is also an aspheric surface such as an expensive cylindrical lens. It is possible to use only an inexpensive spherical lens without using any element. In the case of this measuring method, the length of the measuring light passing through the sample liquid 3, that is, the optical path length is a length d as shown in FIG. 1E. It is difficult to accurately reproduce the optical path length d because a means such as inhalation of a sample by capillary action and cutting of a capillary is used. However, for example, in a qualitative analysis in which an outline of an absorbance spectrum is known, such a degree of variation does not pose a serious problem in practical use. By measuring the absorbance at or above the wavelength and performing an operation to correct the optical path length error from the result, accurate quantification can be performed.

[0019]

According to the present invention, the problems of the prior art, such as the limitation of the liquid volume, the difficulty of injecting / collecting the sample, and the difficulty of washing the cell, are solved, and the absorbance of a very small amount of liquid is solved. Can be measured, and the container for containing the liquid can be manufactured at a low cost. Therefore, it can be disposable, the preparation for measurement is reduced, and the measurement capability is improved.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of an embodiment of the present invention.

FIG. 2 is a side view of an example of the thin tube in the embodiment.

FIG. 3 is a perspective view of an example of a thin tube setting jig in the embodiment.

FIG. 4 is a perspective view of an example of a capillary fixing member in the embodiment.

FIG. 5 is a sectional view of an example of the capillary fixing member in the embodiment.

FIG. 6 is a perspective view of an example of a method for fixing a thin tube in the embodiment.

FIG. 7 is a perspective view of another example of the method for fixing the thin tube in the above embodiment.

FIG. 8 is an explanatory view of a first conventional example.

FIG. 9 is a perspective view of an example of a holder of the first conventional example.

FIG. 10 is an explanatory view of a second conventional example.

FIG. 11 is a perspective view of an example of a holder of the second conventional example.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Thin tube 2 Sample container 3 Sample liquid 4 Thin tube fragment 5 Flux stop

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 21/00-21/61

Claims (2)

(57) [Claims] A liquid to be measured is sucked and held from one end of the capillary by capillary action or suction means. In this state, the capillary is drawn from the suction side end of the capillary so as to include a portion holding the liquid to be measured. Cutting and supporting and fixing the cut portion of the thin tube holding the liquid to be measured to the measuring part of the spectrophotometer so that the central axis thereof coincides with the measuring optical axis, and transmitting the measuring light. Method for measuring the spectral characteristics of trace liquid samples. And a fixing means for positioning and fixing a tip portion of said thin tube holding a liquid to be measured therein, and a tip portion of said thin tube formed by a score line. A device for measuring a minute amount of liquid sample, comprising: means for breaking off.