JP2007132873A - Analytical method, separation method, mixer, and analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical method of a liquid chromatography using a mixer being manufactured and worked easily, which can make the mixer compact by using a simple structure and realize highly reproducible measurement values. <P>SOLUTION: A sample SA is sent with an eluant SS and brought to pass through both a mixer 25 and an auxiliary mixer 27. When the sample SA passes through the mixer 25, the sample SA is sufficiently mixed with the eluant SS especially in a tubular member 51, whereby an almost homogeneous mixed liquid M is obtained. Since the mixer 25 is the tubular member 51 having a built-in spiral 51d, the mixed liquid M is agitated efficiently, and one end 29a of a separation column 29 is filled with the mixed liquid M in stability, thereby improving reproducibility of peak shapes and positions. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an analysis method and a separation method using liquid chromatography, and a mixer and an analysis apparatus used therefor, and more specifically, a gradient type analysis method, a separation method, and the like for performing analysis by changing the composition of an eluent. About.

  As an analysis method using liquid chromatography, there is a method called gradient analysis in which component composition and component analysis are performed by changing the composition of the eluent over time. In such a gradient analysis, by changing the composition of the eluent constituting the moving bed over time, the target component in the sample can be quickly and reliably separated and quantified.

  On the other hand, even if such a gradient analysis is used, the peak shape of a specific test object may be very poor, or the peak shape may change over time, and accurate analysis may be difficult. In order to prevent such a phenomenon, it has been proposed to diffuse the sample by injecting the sample into the eluent and then mixing them with a mixer consisting of tubes that are periodically crushed from different directions (Patent Literature). 1).

  Further, as a liquid chromatography mixer, there is a combination of a first mixer in which a stirring bar rotates and a second mixer having an inner cylinder (see Patent Document 2). Here, the second mixer is obtained by inserting an inner cylinder into the mixer tube, and the mixed solution is spiral along a passage defined by the lead groove on the inner surface of the mixer tube and the peripheral surface of the inner cylinder or the lead groove. Move to.

In addition, as a static mixer used for the analysis of petroleum etc., two opposing components are provided in the pipe, the fluid in the pipe is divided into two, and the axes parallel to the direction of fluid flow are opposite to each other. There exists what deflects two divided fluids so as to rotate (see Patent Document 3).
JP 2003-270226 A JP 9-325141 A Japanese National Patent Publication No. 10-506326

  However, among the above background technologies, when a mixer consisting of tubes crushed from different directions is used, subtle differences in tube crushing, direction, spacing, etc. affect the peak shape, and the reproducibility of measured values May be lower.

  In addition, when a second mixer having an inner cylinder inserted into the mixer tube is used, the mixer becomes larger from the viewpoint of fixing a relatively large inner cylinder in the tube or securing a flow path between the inner cylinder and the tube. The structure becomes complicated and the cost increases.

  Further, in the case of a static mixer provided with two components that form a flow in a pipe, the shape of the components fixed in the pipe becomes complicated due to the formation of flows that rotate in opposite directions. Processing becomes difficult.

  Therefore, the present invention provides a liquid chromatography analysis method and separation method using a mixer that has high reproducibility of measurement values, can be downsized with a simple structure, and is easy to manufacture and process. Objective.

  Another object of the present invention is to provide a mixer and an analysis apparatus suitable for realizing a gradient type liquid chromatography analysis method and the like that can accurately separate and detect adjacent peaks.

  In order to solve the above-described problems, an analysis method according to the present invention is an analysis method for analyzing a component of a sample while changing the composition of the eluent by using (a) liquid chromatography, and (b) elution A step of injecting the sample into the liquid and diffusing the sample into the eluent while mixing the sample and the eluent using a tubular member having a partition having a shape obtained by twisting a plate-like member; (c ) A step of filling one end of the separation column with a mixed solution obtained by diffusing the sample in the eluent, and (d) an elution discharged from the other end of the separation column while supplying the mixed solution or the eluent to one end of the separation column. And sequentially detecting components of the liquid.

  In the above analysis method, the sample is diffused into the eluent in the ground state using a tubular member before supplying the sample or the like to one end of the separation column. The portion is filled in a stable state. At this time, since the tubular member incorporates a partition in the shape of a plate-shaped member, a natural spiral flow can be formed on the front side and the back side of the partition, respectively, and the mixed solution is reproducible in the eluent. It can be diffused well and uniformly. Therefore, not only can the sample solvent behave like a moving bed in the separation column, but it can also accurately separate and detect components with adjacent peaks with high reproducibility in analysis using gradient liquid chromatography. be able to.

  In a specific aspect or aspect of the present invention, in the analysis method, the tubular member includes a straight pipe having a circular cross-section passage and holding a partition in the passage, and the partition is an axial direction of the straight pipe. And has a circular outline substantially matching the circular cross section of the passage. In this case, it is possible to cause sufficient swirling of the mixed liquid in the mixing tubular member, and efficient stirring becomes possible.

  In another aspect of the present invention, the partition is formed of an elongated belt-like member, and is constant around a center line substantially along the longitudinal direction of the belt-like member from one end to the other end along the axial direction of the straight pipe. And a shape twisted by 180 ° or more. In this case, it is possible to form a smooth flow of the mixed solution or the eluent in the straight pipe of the tubular member with almost no hindrance to the flow of the mixed solution. Can be separated.

  In still another aspect of the present invention, the plate-like member has a width in a direction orthogonal to the twisting direction at least partially and wider than the inner diameter of the straight pipe, and is sandwiched by the inner face of the straight pipe at least in part. To be fixed. In this case, the partition can be fixed easily and reliably in the straight pipe, and the structure of the tubular member can be simplified.

  In yet another aspect of the present invention, the eluent into which the sample has been injected is supplied to the tubular member and then passed through an extension tube having a predetermined length or more. In this case, the sample and the eluent can be mixed and stirred more efficiently, and the ability to separate and detect components having adjacent peaks can be further enhanced.

  The separation method according to the present invention is a separation method for separating a sample into components while changing the composition of the eluent by using (a) liquid chromatography, and (b) injecting the sample into the eluent. And a step of diffusing the sample into the eluent while mixing the sample and the eluent using a tubular member having a partition having a shape obtained by twisting a plate-like member, and (c) the sample in the eluent And (d) discharging the eluate from the other end of the separation column while supplying the mixed liquid or the eluent to one end of the separation column. .

  Even in the above separation method, the sample is diffused into the eluent in the ground state using the tubular member before supplying the sample or the like to one end of the separation column. It is possible to accurately separate and detect a component having a close peak in a stable state and having a close peak with high reproducibility.

  In addition, the mixer according to the present invention includes (a) an inlet connected to an injection device side for injecting a sample into an eluent for liquid chromatography, and (b) a partition having a twisted shape of a plate-like member. And a tubular member that allows the mixture of the sample and the eluent to pass therethrough, and (c) an outlet connected to the separation column side for separating the sample according to the components while allowing the mixture or eluent to pass through. With.

  In the above mixer, the tubular member through which the liquid mixture passes has a built-in partition having a twisted plate-like member, so that the eluent and the sample can be mixed and stirred efficiently before filling the sample into one end of the separation column. can do. Since the liquid mixture obtained by such agitation is filled in a stable state at the inlet of the separation column, for example, in the analysis using a liquid chromatography such as a gradient type, components having adjacent peaks are highly reproducible. It can be separated and detected accurately.

  In a specific aspect of the present invention, in the mixer, the tubular member includes a straight pipe having a circular cross-section passage and holding a partition in the passage, and the partition is viewed from the axial direction of the straight pipe. It has a circular contour that substantially matches the circular cross section of the passage.

  In a more specific aspect of the present invention, the plate-like member has a width in a direction orthogonal to the twisting direction at least partly wider than the inner diameter of the straight pipe, and at least partly on the inner surface of the straight pipe. It is clamped and fixed.

  The analyzer according to the present invention includes (a) an injection device for injecting a sample into an eluent for liquid chromatography, and (b) a shape that is connected to the outlet side of the injection device and twists a plate-like member. (C) a separation column that is connected to the outlet side of the tubular member and separates the sample according to the component while allowing the eluent charged at one end together with the sample to pass through; Detection means for sequentially detecting the components of the eluate discharged from the other end of the separation column.

  In the analyzer, since the tubular member through which the mixed solution passes incorporates a partition having a shape obtained by twisting a plate-like member, the mixed solution obtained by stirring using the tubular member is stabilized at the inlet of the separation column. In analysis using liquid chromatography such as a gradient type packed in a state, components having adjacent peaks can be accurately separated and detected with high reproducibility.

[First Embodiment]
FIG. 1 is a block diagram conceptually illustrating an analyzer for carrying out an analysis method according to the first embodiment of the present invention.

  The illustrated analyzer 10 is a gradient-type high performance liquid chromatography device, and can perform component separation and component analysis by changing the composition of the eluent over time. This analyzer 10 is mixed with a plurality of different solvents A and B at a desired ratio to generate an eluent SS, and connected to the outlet side of the eluent supplier 21 to be connected to the eluent SS. A sample injection device 23 for injecting a predetermined amount of the sample SA to be analyzed, a mixer 25 connected to the outlet side of the sample injection device 23 to uniformly mix the sample SA and the eluent SS, and an outlet side of the mixer 25 An auxiliary mixer 27 that is connected and performs auxiliary mixing, and a separation column 29 into which the mixed solution M and the like that have passed through the auxiliary mixer 27 are injected from one end 29a. Furthermore, the analyzer 10 is connected to the other end 29b of the separation column 29 through a pipe and detects the fluorescence and absorbance of each component separated from the sample SA, and the fluorescence detected by the detector 31 and An arithmetic processing unit 33 that analyzes a signal corresponding to light absorption and measures the concentration of the separated component and the like, and a tank 35 that stores the eluate S that has passed through the detector unit 31 are provided.

  The eluent supply device 21 has a supply source of the solvents A and B and a pump for pumping, and emits an eluent SS in which the solvents A and B are mixed at a desired ratio at a high pressure. Further, the eluent supply device 21 can appropriately change the mixing ratio of the solvents A and B, and the composition ratio of the eluent SS supplied to the separation column 29 after the sample SA is filled in the separation column 29 can be changed continuously or It can be changed in stages. Thereby, the gradient analysis of the sample SA becomes possible. The solvents A and B can be organic solvents such as water and acetonitrile.

  The sample injection device 23 includes a manual injector or an autosampler, and can inject a desired amount of sample SA into the eluent SS supplied from the eluent supply device 21 at a desired timing. The amount of the sample SA injected into the eluent SS can be appropriately changed in consideration of other measurement conditions and the like. The sample SA is obtained by dissolving a substance to be analyzed (test object) in a sample solvent such as acetonitrile.

  The mixer 25 is composed of a tubular member (to be described in detail later) containing a partition having a shape obtained by twisting a plate-like member, and the eluent SS and the sample SA are separated by a spiral flow formed in the tubular member. Uniform mixing, that is, uniform diffusion of the sample SA injected into the eluent SS is achieved, and the mixture M having a substantially uniform composition is supplied to one end 29 a of the separation column 29. The mixer 25 constitutes a diffusion means together with an auxiliary mixer 27 described later.

  FIG. 2 is a partially broken side view for explaining the structure of the mixer 25. 3 is an enlarged cross-sectional view of a tubular member that is a main part of the mixer 25 of FIG. 2, and FIGS. 4 (a) and 4 (b) show the partition inserted in the tubular member shown in FIG. It is the side view and end view explaining a shape.

  As shown in FIG. 2, the mixer 25 includes a tubular member 51 that forms a spiral flow in the tube and stirs the mixed liquid M and the like, and first and second fasteners 53 provided at both ends of the tubular member 51. , 55.

  Here, as shown in FIG. 3, the tubular member 51 includes a stainless steel tube 51 a that is an elongated straight pipe and a spiral 51 d that is a partition housed in the stainless steel tube 51 a. The spiral 51d has a length of about 1/5 or less of the stainless steel tube 51a, and is fixed to one end 51b on the inlet side of the stainless steel tube 51a. Here, as a specific example of the stainless steel tube 51a, an inner diameter of 0.04 inch (about 1 mm) was used. In a specific example, the axial length a of the stainless steel tube 51a is, for example, 50 mm to 55 mm, and the pushing amount b corresponding to the distance from the end of the stainless steel tube 51a to the end of the spiral 51d is, for example, 0. .3 mm to 0.5 mm. The stainless tube 51a is not limited to stainless steel, and may be formed of other materials as long as the spiral 51d can be stably fixed and has corrosion resistance against the mixed solution M or the like. You can also.

  The first fastener 53 is an inlet portion composed of a union joint for connecting a stainless tube, and a union main body 53a having a shape in which two hexagonal column portions each having an internal thread formed therein are arranged in series, and a tip of a ferrule shape. And a pair of screw members 53b and 53c. When assembling, with one end 51b of the stainless steel tube 51a inserted into the center hole 53f of the screw member 53b, the screw member 53b is screwed into the union main body 53a from one side and tightened, and upstream of the center hole of the screw member 53c. With the stainless steel tube 61 inserted, the screw member 53c is screwed into the union main body 53a from the other side and tightened. Thereby, the end 51b of the stainless steel tube 51a is liquid-tightly connected to the tube 61. In a specific example, as the tube 61, a stainless tube or a PEEK tube was used. These tubes have, for example, an inner diameter of 0.01 inch and an outer diameter of 1/16 (about 1 mm), and the length to the sample injection device 23 in FIG. 1 is 1000 mm.

  On the other hand, the 2nd fastener 55 is an exit part which consists of a union joint similar to the 1st fastener 53, and is provided with the union main body 55a and a pair of screw member 55b, 55c. When assembling, with the other end 51c of the stainless steel tube 51a inserted into the central hole 55f of the screw member 55b, the screw member 55b is screwed into the union main body 53a from one side and tightened, and downstream piping is connected to the central hole of the screw member 53c. With the stainless steel tube 62 inserted, the screw member 55c is screwed into the union main body 55a from the other side and tightened. Thereby, the other end 51c of the stainless steel tube 51a is liquid-tightly connected to the tube 62. In a specific example, as the tube 62, a stainless steel tube or a PEEK tube was used. These tubes had, for example, an inner diameter of 0.01 inch and an outer diameter of 1/16 (about 1 mm), and the length to the separation column 29 in FIG. 1 was 500 to 1000 mm. The tube 62 is wound many times in the shape of a coil spring, and constitutes the auxiliary mixer 27 of FIG.

  As shown in FIGS. 4 (a) and 4 (b), the spiral 51d has a shape obtained by twisting the belt-like member one and a half times (540 °), and when viewed from the side, the width periodically increases and decreases from the end surface. When viewed, it has a circular outline.

  FIG. 4C is a diagram for explaining a band-like member 71 that is a material of the spiral 51d. The band-shaped member 71 is a plate material whose width decreases from one end to the other end, and is obtained by cutting a metal material into a strip shape so as to have a width larger at least at one end than the inner diameter of the stainless tube 51a. After the cutting, the outer periphery of the belt-like member 71 is polished, and using a suitable tool, the belt-like member 71 is twisted at a constant rate around the center line CL substantially along the longitudinal direction to obtain a long spiral material. This spiral material is cut at an appropriate location to obtain a spiral 51d shown in FIG. In a specific example, a titanium foil plate was used as the band-like member 71. This titanium foil plate was prepared, for example, having a width d at one end of about 1 mm, a width e ′ at the other end of about 0.5 mm, and a total length f ′ of 50 mm. The resulting spiral 51d of the example had a width d at one end of about 1 mm, a width e at the other end of about 0.8 to 0.9 mm, and a total length f of 5 mm. As a result, the distance g that the spiral 51d is semi-twisted is about 1.5 to 2 mm.

  In the spiral 51d described above, the width in the direction orthogonal to the twisting direction is wider than the inner diameter of the stainless tube 51a at the one end S1, and the direction orthogonal to the twisting direction is higher than the inner diameter of the stainless tube 51a at the other end S2. The width is narrow. As a result, one end S1 of the spiral 51d fits smoothly in the stainless tube 51a, but the other end S2 of the spiral 51d is securely fixed in a state of being compression-fitted in the stainless tube 51a. If it is desired to more securely fix the spiral 51d, processing / deformation such as reducing the inner diameter of the stainless tube 51a can be performed on the downstream side of the spiral 51d.

  The spiral 51d has a front surface FF and a back surface RF. That is, since the spiral 51d fixed in the stainless tube 51a divides the flow path in the stainless tube 51a into two, two spiral flows corresponding to the front surface FF and the back surface RF, that is, the spiral flows SF and SR are forced in the tube. (See FIG. 3). Therefore, the mixed solution M and the eluent SS passing through the stainless steel tube 51a, that is, the tubular member 51 are efficiently stirred as two spiral flows SF and SR by passing through the spiral 51d. The stirring state and the mixed state become extremely stable.

  Returning to FIG. 1, the auxiliary mixer 27 is an extension tube obtained by processing the tube 62 extending from the tubular member 51 into a coil spring shape, as already described. The auxiliary mixer 27 can mix the sample SA while gradually diffusing the sample SA in the eluent SS, for example, by extending the flow path of the mixed solution M and the eluent SS, thereby further stabilizing the stirring by the mixer 25. Have a role.

  The separation column 29 is made of a tubular member and encloses silica gel or the like as a stationary phase for sample separation. An oven (not shown) with a built-in heater for adjusting temperature is provided around the separation column 29, so that the temperature inside the separation column 29 can be kept constant.

  In addition, the detector unit 31 includes a glass pipe or the like through which the eluate S discharged from the other end 29b of the separation column 29 passes as detection means. The detector 31 detects components separated by an optical method such as detecting the fluorescence by irradiating the eluent S passing therethrough with ultraviolet light and detecting the absorbance of infrared rays.

  The arithmetic processing unit 33 performs appropriate processing on the signal corresponding to the fluorescence and absorbance output from the detector unit 31 to correlate the concentration of the separated component contained in the eluate S and the numerical value corresponding to the retention time. Record and display as

  The tank 35 is for recovering the eluate S which has been analyzed by the detector unit 31. If the tank 35 is switched in consideration of the retention time, the eluate S can be accurately separated for each component. it can.

  Hereinafter, the operation of the analyzer 10 shown in FIG. 1 will be described. First, the eluent supply device 21 is operated to introduce the eluent SS corresponding to the ground state in the gradient analysis into the sample injection device 23 and further to the separation column 29 at a constant flow rate. For example, when the solvents A and B are water and acetonitrile, water is the main component of the ground state eluent SS.

  Thereafter, the sample SA is injected into the eluent SS through the sample injection device 23 at an appropriate timing by a desired amount (for example, 2 μL). Here, the sample SA is prepared by, for example, dissolving the test object in a sample solvent made of only acetonitrile. In this way, the sample SA is adjusted with a sample solvent made only of acetonitrile in consideration of the case where the test object included in the sample SA is susceptible to decomposition by water. This is because the analysis conditions are defined in detail in the standard document regarding the adjustment method of the sample SA, the sample injection amount, etc., and these cannot be easily changed. Therefore, when the type of detection target and detection conditions permit, the sample SA can be adjusted with various solvents other than acetonitrile.

  By the injection as described above, the sample SA is sent to the eluent SS and passes through the mixer 25 and the auxiliary mixer 27. When passing through the mixer 25, the sample SA is sufficiently mixed with the eluent SS, particularly in the tubular member 51, and a substantially uniform mixed solution M is obtained. Further, when passing through the auxiliary mixer 27, the mixed solution M becomes more uniform and stable. Here, the mixed solution M is obtained by mixing, for example, a sample SA obtained by dissolving a test object in a sample solvent made of only acetonitrile and an eluent SS made of substantially water, By using such a mixer 25 or the like, the test object can be brought into a state close to a case where the test object is dissolved in a solvent of water: acetonitrile = 1: 1, for example.

  The mixed solution M emitted from the mixer 25 is then pushed out to the eluent SS fed from the sample injection device 23 and introduced into one end 29 a of the separation column 29. Thereby, the filling of the sample SA into the separation column 29 is completed. In addition, since the eluent SS itself for sending the mixed liquid M is sufficiently stirred by the mixer 25 and the like, the eluent SS is kept uniform and stable.

  After that, by continuing to supply the eluent SS from the eluent supply device 21 to the separation column 29, the mixed liquid M, that is, the sample SA filled in the one end 29a of the separation column 29 is gradually separated for each component. It moves to the other end 29b of the column 29. By such movement, the sample SA is separated into bands for each component in the separation column 29, and a specific standard substance and its related substances can be accurately separated. At this time, the composition of the eluent SS is gradually changed for gradient analysis. For example, the concentration of water in the eluent SS is decreased and the concentration of acetonitrile is increased. Thereby, since the separation conditions of the sample SA can be controlled with a certain degree of arbitraryness, it is possible to obtain a quick and highly accurate measurement result. That is, when the polarity difference of the solute to be tested is large, a highly polar compound that is difficult to be retained in the separation column 29 is separated with a separation liquid that initially contains a large amount of water, and then the ratio of acetonitrile is gradually increased to make it relatively polar. Can be separated.

  When the diffusion / dilution by the mixer 25 and the auxiliary mixer 27 is excessive, not only is the concentration of the sample SA filled in the one end 29a of the separation column 29 diluted, but the mixture M has a wide band. As a result, the bandwidth of the separation peak due to the separation column 29 widens, and the accuracy of component separation may decrease instead. Therefore, the size of the mixer 25 and the auxiliary mixer 27 and the internal structure are adjusted, and the sample SA and the eluent SS are mixed within a range in which the diffusion and dilution by these are not excessive.

  On the other hand, when there is no diffusion / dilution by the mixer 25 and the auxiliary mixer 27, the separation column 29 in the ground state and stable with the eluent SS having a composition close to water is used only with an organic solvent such as acetonitrile. The adjusted sample SA is filled. In this case, the sample SA packed in the one end 29a of the separation column 29 behaves as if it is a mobile phase, that is, an eluent SS. That is, the solute that should be concentrated at one end 29a of the separation column 29, that is, the test substance moves so as to be pushed out together with the organic solvent band such as acetonitrile in the separation column 29, and the elution of each component is accelerated. It becomes difficult to separate a test object (for example, a related substance of a specific type of standard substance) containing a component having a short retention time.

  On the other hand, when the mixer 25 and the auxiliary mixer 27 as in the above embodiment are arranged close to the one end 29a of the separation column 29, the sharpness of each peak shape obtained when separating a component having low polarity from the sample SA. Is improved. Moreover, since the mixer 25 is the tubular member 51 containing the spiral 51d, the mixed solution M is efficiently stirred and filled in the one end 29a of the separation column 29 in a stable state, and the reproducibility of the peak shape and position is improved.

  Specific examples will be described below. The analyzer 10 has a structure as shown in FIG. The shape and dimensions of the tubular member 51 constituting the mixer 25 are the same as those described in the embodiment in FIGS. As the tube 61, a stainless steel tube having an inner diameter of 0.01 inch and a length of 1000 mm was used, and as the tube 62, a stainless steel tube having an inner diameter of 0.01 inch and a length of 1000 mm was used.

  5 to 7 are graphs for explaining the effects of the mixer 25 and the auxiliary mixer 27 in the analysis of a related substance of a certain standard substance. FIG. 5 shows the result of analyzing the related substance A by the analyzer 10 of the example. Although the waveforms of the first to fourth analysis results are arranged in the vertical direction, it can be seen that the peak shape and position of the related substance A are the same in all the waveforms. FIG. 6A is a graph obtained by enlarging the graph of FIG. 5 in the horizontal direction, and it can be confirmed that the peak shape and position of the related substance A are exactly the same. On the other hand, FIG. 6B shows the result of analyzing the related substance A by mixing the sample with a conventional mixer (see Japanese Patent Application Laid-Open No. 2003-270226), which is similar to the examples. It can be confirmed that the peak shape and position of A are fluctuating.

  FIG. 7A is a chromatogram for explaining the analysis result of the sample using the analyzer 10 of the embodiment including the mixer 25 and the like, and FIG. 7B is an analysis of the comparative example not including the mixer 25 and the like. It is a chromatogram explaining the analysis result of the same substance using an apparatus. When the analyzer 10 of the example of FIG. 7A is used, the peak of the related substance A is detected as a peak having a large rising angle. However, when the analyzer of the comparative example is used, the peak of the related substance A is detected. The rising angle of the peak is small and it is detected as a peak with intense reading. Moreover, although it does not understand from a graph, when the analysis apparatus of the comparative example was used, the detection of the peak of the related substance A was comparatively unstable. Further, when the analyzer 10 of the example is used, the peak of the isomer immediately before the standard substance (large peak on the right side of the center) which is the sample SA is separated from the peak of the sample SA. When the analyzer is used, the peak of the isomer just before the sample SA overlaps the peak of the sample SA.

[Second Embodiment]
Hereinafter, an analysis apparatus for performing the analysis method and the like according to the second embodiment will be described. The analysis method of the second embodiment is a modification of the mixer 25 used in the analysis method of the first embodiment, and only the improved mixer will be described here.

  FIG. 8 is a cross-sectional view for explaining the external appearance of the mixer of the present embodiment, and corresponds to FIG. 3 of the first embodiment. In this case, the tubular member 151 provided in the mixer has a plurality of spirals 51d and 151d fixed in the stainless tube 51a. Both spirals 51d and 151d have the same shape, and are sequentially inserted into the stainless tube 51a and fixed. Note that the illustrated spirals 51d and 151d are examples, and the length and the amount of twist can be different from the illustrated shapes.

[Third Embodiment]
Hereinafter, an analysis apparatus for performing the analysis method and the like according to the third embodiment will be described. In the analysis method and the like of the third embodiment, the mixer 25 used in the analysis method and the like of the first embodiment is changed as in the case of the second embodiment.

  FIG. 9 is a cross-sectional view for explaining the appearance of the mixer of the present embodiment, and corresponds to FIG. 3 of the first embodiment. In this case, the tubular member 251 provided in the mixer fixes a spiral 251d having a complex shape in the stainless tube 51a. As shown in FIG. 10, the spiral 251d is obtained by dividing the belt-like member 271 into two parts excluding the connecting portion 271a, and twisting the two strip portions 271b and 271c into spiral portions 251h and 251i, respectively.

  Although the present invention has been described based on the above embodiments, the present invention is not limited to the above embodiments. For example, the shapes and dimensions of the tubular members 51, 151, and 251 included in the mixer 25 can be changed as appropriate according to the application. In particular, the length of the spiral 51d incorporated in the tubular member 51, the number of twists per unit distance when forming the spiral 51d, the total number of twists, and the like can be appropriately changed.

  In the above embodiment, the spiral 51d to be incorporated into the tubular member 51 or the like has a shape twisted at a constant ratio around the center line CL, but the spiral 51d has a part that is not twisted. A portion where the ratio of twist and twist changes can be provided.

  In the above-described embodiment, the spiral 51d incorporated in the tubular member 51 or the like is composed of a strip-like strip-shaped member 71. However, the strip-shaped member 71 can be a member having a narrow lateral width except at both ends. Further, one or more openings can be formed at appropriate positions of the belt-like member 71. In such a case, the flow path in the stainless steel tube 51a is not completely partitioned by the front surface FF and the back surface RF.

  Moreover, in the said embodiment, although the tubular member 51 is incorporated in the mixer 25 using the union-like fasteners 53 and 55, as long as the flow of the liquid mixture M etc. is not prevented, various shapes of fitting apparatuses should be utilized. Can do.

  In the above embodiment, the case where the sample SA is adjusted with acetonitrile has been described. However, even if the sample is adjusted with another solvent, the same effect as in the above embodiment can be obtained as long as the sample SA has affinity with the eluent SS. It is done. Similarly, the type of eluent SS and the rate of composition change in gradient analysis can be changed as appropriate according to the purpose and application.

It is a block diagram which illustrates notionally the analyzer for implementing the analysis method which concerns on 1st Embodiment. It is a partially broken side view explaining the structure of a mixer. It is a figure explaining the longitudinal cross-sectional structure of the tubular member which comprises the mixer of FIG. (A) is a side view of a spiral incorporated in the mixer, (b) is an end view of the spiral, and (c) is a diagram for explaining a band-shaped member that is a material of the spiral. The result of having analyzed the related substance by the analyzer of an Example is shown. (A) is the graph which expanded the graph of FIG. 5 to the horizontal direction, (b) shows the result of having analyzed the related substance by the conventional mixer. (A) is a graph explaining the analysis result using the analyzer of an Example, (b) is a graph explaining the analysis result using the analyzer of the comparative example which is not provided with a mixer. It is a figure explaining the mixer integrated in the analyzer which concerns on 2nd Embodiment. It is a figure explaining the mixer integrated in the analyzer which concerns on 3rd Embodiment. It is a figure explaining preparation of the mixer of FIG.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Analyzing device, 21 ... Eluent supply device, 23 ... Sample injection device, 25 ... Mixer, 51 ... Tubular member, 51d ... Spiral, 27 ... Auxiliary mixer, 29 ... Separation column, 29a ... One end, 29b ... Other end, 31 ... Detector unit, 33 ... Arithmetic processing unit, 35 ... Tank, A, B ... Solvent, M ... Mixed solution, S ... Eluate, SA ... Sample, SS ... Eluent

Claims (10)

An analysis method for analyzing components of a sample while changing the composition of an eluent by using liquid chromatography,
The sample is injected into the eluent, and the sample is diffused into the eluent while mixing the sample and the eluent using a tubular member having a partition having a shape obtained by twisting a plate-like member. Process,
Filling one end of a separation column with a mixed solution obtained by diffusing the sample in the eluent;
And a step of sequentially detecting the components of the eluate discharged from the other end of the separation column while supplying the mixed solution or the eluent to one end of the separation column.   The tubular member includes a straight pipe having a passage having a circular cross section and holding the partition in the passage, and the partition substantially coincides with the circular cross section of the passage when viewed from the axial direction of the straight pipe. The analysis method according to claim 1, wherein the analysis method has a circular outline.   The partition is formed of an elongated belt-like member, and is 180 ° or more at a constant rate around a center line substantially along the longitudinal direction of the belt-like member from one end to the other end along the axial direction of the straight pipe. The analysis method according to claim 2, wherein the analysis method has a twisted shape.   The plate-like member has a width in a direction perpendicular to the twisting direction at least partially and wider than the inner diameter of the straight pipe, and is clamped and fixed to the inner surface of the straight pipe at least in part. The analysis method according to any one of claims 2 and 3, wherein:   The analysis method according to any one of claims 1 to 4, wherein the eluent injected with the sample is supplied to the tubular member and then passed through an extension tube having a predetermined length or more. A separation method for separating a sample into components while changing the composition of the eluent by using liquid chromatography,
The sample is injected into the eluent, and the sample is diffused into the eluent while mixing the sample and the eluent using a tubular member having a partition having a shape obtained by twisting a plate-like member. Process,
Filling one end of a separation column with a mixed solution obtained by diffusing the sample in the eluent;
And a step of discharging the eluent from the other end of the separation column while supplying the mixed liquid or the eluent to one end of the separation column. An inlet connected to an injection device for injecting a sample into an eluent for liquid chromatography;
A tubular member that incorporates a partition having a shape obtained by twisting a plate-like member, and allows a mixed solution of the sample and the eluent to pass through;
A mixer comprising: an outlet connected to a separation column side for separating the sample according to the component while allowing the mixed solution or the eluent to pass therethrough.   The tubular member includes a straight pipe having a passage having a circular cross section and holding the partition in the passage, and the partition substantially coincides with the circular cross section of the passage when viewed from the axial direction of the straight pipe. 8. The mixer according to claim 7, wherein the mixer has a circular outline.   The plate-like member has a width in a direction perpendicular to the twisting direction at least partially and wider than the inner diameter of the straight pipe, and is clamped and fixed to the inner surface of the straight pipe at least in part. The mixer according to claim 8. An injection device for injecting a sample into an eluent for liquid chromatography;
A tubular member that is connected to the outlet side of the injection device and incorporates a partition having a shape obtained by twisting a plate-like member;
A separation column that is connected to the outlet side of the tubular member and separates the sample according to the components while allowing the eluent packed at one end together with the sample to pass through;
An analysis apparatus comprising: a detection unit that sequentially detects components of the eluate discharged from the other end of the separation column.

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