Preparative chromatography support device, preparative chromatography apparatus, and preparative chromatography method

Abstract

To find a method capable of using a result of thin layer chromatography or column chromatography to predict a result of liquid chromatography with high accuracy for a wide range of compounds, and setting an optimal separation condition.SOLUTION: A chromatography support device for fractionation comprising calculation formula acquisition means is a device for providing information on fractionation in column chromatography based on a measured value of an Rf value when using a mixed solvent with a mixture ratio B of two specific solvents measured by thin layer chromatography or liquid chromatography. The calculation formula acquisition means acquires a1 in the relational expression Rf=a1B+b1 (1) or a2 in the relational expression logk'=a2logB+b2 (2), (where a retention ratio is defined as k'=(tR-t0) / t0 (3), a solvent ratio is defined as B), based on B and Rf, which are measured values of one measurement result in TLC.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a preparative chromatography support apparatus, a preparative chromatograph apparatus, and a preparative chromatography method. [Background technology]

[0002] In liquid chromatography, a solution containing the dissolved sample is passed through a stationary phase packed into a column. At this time, the components of the sample introduced into the column pass through the column at different times due to their interactions with the stationary phase and affinity for the mobile phase. Therefore, by the time the sample is discharged from the column, the components are separated. Substances that have strong interactions with the stationary phase or weak affinity for the mobile phase remain in the column longer and are discharged from the column more slowly.

[0003] In this type of liquid chromatography, the selection of solvent and column size is crucial, and the elution time of the sample is closely related to these conditions. Therefore, being able to predict the test results before actually performing liquid chromatography can improve the efficiency of the experiment.

[0004] There is also thin-layer chromatography (TLC), which is performed using the same principle as liquid chromatography described above. In thin-layer chromatography, the sample is dropped onto a thin layer of material used as the stationary phase in a column, and this thin layer is immersed in the eluent, which acts as the mobile phase. Then, due to capillary action, the eluent is drawn up to the thin layer, and the sample is also drawn up, resulting in the mobility R of the sample relative to the eluent. f This allows us to determine (Figures 4 and 5). In this way, the mobility R obtained by TLC performed with an eluent of any solvent ratio can be determined. f It is known that there is a correlation between this and the elution time in liquid chromatography.

[0005] The applicant has filed Patent Documents 1 to 4 concerning a liquid chromatography control device that utilizes the correlation between TLC results and the resolution of liquid chromatography to predict liquid chromatography results in advance based on TLC results, thereby enabling efficient liquid chromatography.

[0006] In Patent Document 1, mobility R f An approximate treatment based on a linear function is performed to determine the relationship between the mixing ratio (B / A) of the solvent and the preparative chromatography (TLC). This approximation is extremely useful industrially because it allows for the prediction of liquid chromatography results from TLC results. However, the accuracy between the predicted preparative chromatography results and the actual preparative results is desirable as much as possible, and higher accuracy allows for the setting of optimal conditions for preparative liquid chromatography. Therefore, a preparative chromatography support system that can achieve higher accuracy would be preferable.

[0007] Considering these points, the inventors of the present invention investigated a system that further enhances the functionality of liquid chromatography, predicts elution time with greater precision than that specified in Patent Document 1, and enables the setting of conditions for optimal separation at all times. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2003-240765 [Patent Document 2] Japanese Patent Publication No. 2017-32403 [Patent Document 3] Japanese Patent Publication No. 2017-125686 [Patent Document 4] Japanese Patent Publication No. 2007-3398 [Overview of the project] [Problems that the invention aims to solve]

[0009] In view of the above, the present invention aims to find a method that allows for the highly accurate prediction of liquid chromatography results for a wide range of compounds using the results of thin-layer chromatography or column chromatography, and to set optimal separation conditions. In other words, the objective is to provide a liquid chromatography method that enables efficient liquid chromatography under optimal conditions at all times. [Means for solving the problem]

[0010] The present invention is a preparative chromatography support device equipped with a means for acquiring calculation formulas, This device provides information regarding preparative chromatography in column chromatography based on measured Rf values ​​when using a mixed solvent of two specific solvents at a mixing ratio B, as measured by thin-layer chromatography or liquid chromatography. The means of obtaining the calculation formula is, Rf = a1B + b1(1) a1 or in the relational expression log k' = a² log B + b² (2) (however, Retention ratio:k'=(t R -t0) / t0(3) Solvent ratio: B a2 in the relational expression This preparative chromatography support device is characterized by acquiring B and Rf, which are actual measured values ​​from a single measurement in TLC.

[0011] The above-described preparative chromatography support device preferably has a means for measuring the relationship between the Rf value and the solvent mixing ratio B for a large number of compounds for a specific combination of two solvents, and for obtaining a calculation formula based on the data obtained therefrom. The present invention is also a preparative chromatography apparatus characterized by comprising the preparative chromatography support apparatus described above.

[0012] The present invention includes a step (1) of performing thin-layer chromatography of a compound to be fractionated in a mixed solvent with a mixing ratio B of two specific solvents A step (2) of inputting the Rf value and the mixing ratio B obtained in step (1) into the fractionation chromatography support device according to claim 1 Based on the data input in step (2), the fractionation chromatography support device Rf = a1B + b (1) Or logk’ = a2logB + b (2) (However Retention ratio: k’=(t R -t0) / t0 (3) A step (3) of obtaining a1 or a2 in the relational expression Based on the relational expression, a step (4) of determining gradient conditions in fractionation chromatography And [[ID=2-4]]A step (5) of performing chromatography according to the gradient conditions determined in step (4) It is also a fractionation chromatography method characterized by having the above steps

Effect of the Invention

[0013] With the fractionation chromatography support device of the present invention, a more accurate prediction can be made about the results of chromatography, thereby improving the efficiency of the separation operation in chemical experiments

Brief Description of the Drawings

[0014] [Figure 1] It is a diagram showing the relationship between the measured values of the Rf value and the solvent mixing ratio B for many kinds of compounds [Figure 2] In the relational expression shown in FIG. 1, it is a diagram showing the relationship between B and the slope a at a specific Rf value [Figure 3] It is a diagram showing the relationship between the intercept and Rf in the relationship between B and the slope a at a specific Rf value shown in FIG. 2 [Figure 4] It is a diagram for explaining the gradient [Figure 5] This is a perspective view showing the apparatus used for performing TLC (Total Light Curing). [Figure 6] These are schematic diagrams of silica gel thin-layer plates used in TLC, with (a) showing the plate before TLC and (b) showing the plate after TLC. [Figure 7] This is a schematic diagram showing a liquid chromatograph apparatus to which the present invention is applied. [Figure 8] This is a dissolution curve obtained based on the calculation formula acquired by the method of the present invention. [Figure 9] This is a dissolution curve obtained based on a calculation formula acquired using conventional methods. [Modes for carrying out the invention]

[0015] The preparative chromatography support apparatus of the present invention can predict the behavior when preparative chromatography is performed based on the TLC results of the target compound to be separated, find the optimal chromatography conditions, and accurately predict the chromatography results before the work is performed.

[0016] In such preparative chromatography support systems, a method has been employed in which the relationship between the solvent mixing ratio and the Rf value is clarified based on the TLC results, and the chromatographic results are predicted based on this. In Patent Document 1, the prediction was made based on the premise that the relationship between the mixing ratio of each component of the eluent and the mobility Rf of the sample is proportional, and that the rate of change of the sample mobility Rf with respect to the mixing ratio of each component of the eluent is the same even if the type of sample changes.

[0017] Patent Document 2 makes predictions based on a different relational formula than that in Patent Document 1, but it is based on the premise that the rate of change in that relational formula remains the same even if the type of sample changes.

[0018] Patent Document 3 discloses performing TLC at at least two points with different solvent mixing ratios. However, it is preferable that accurate prediction can be achieved by performing TLC at a single point.

[0019] The premise in Patent Documents 1 and 2 that "the rate of change of sample mobility Rf with respect to the mixing ratio of each component of the eluent is the same even if the type of sample changes" is not absolutely correct and should be considered an approximation. In other words, although a certain degree of accuracy can be predicted based on this premise, in reality, the relationship between the mixing ratio of each component of the eluent and the sample mobility Rf undergoes some degree of change when the type of sample changes.

[0020] Therefore, in cases where more accurate analysis is required, it is necessary to perform predictions with higher accuracy than those described in Patent Documents 1 and 2. To address such situations, the object of the present invention is to provide a method for predicting chromatographic results with higher accuracy.

[0021] To illustrate the method of the present invention, Figure 1 shows a graph illustrating the relationship between the solvent mixing ratio B and the Rf value for many compounds, obtained by performing TLC at various solvent mixing ratios. From the results in Figure 1, it is clear that there are differences in the rate of change of the sample's mobility Rf with respect to the mixing ratio of each component of the eluent, depending on the compound.

[0022] On the other hand, this difference is larger between compounds where Rf is high even in nonpolar solvents and compounds where Rf is not high unless a polar solvent is used, that is, between compounds where a straight line exists to the left on the graph in Figure 1 and compounds where a straight line exists to the right. For compounds where the positions of the graphs themselves are close, the difference in the rate of change of the sample's mobility Rf with respect to the mixing ratio of each component of the eluent is a relatively similar value. Furthermore, a similar trend is observed when a similar analysis is performed based on the general formula (2) above.

[0023] Based on this perspective, the present invention was completed. That is, the relationship between the mixing ratio of each component of the eluent and the mobility Rf is Rf = a1B + b1(1) When expressed as such, a1 corresponds to the rate of change of the sample's mobility Rf with respect to the mixing ratio of each component of the eluent. In Patent Document 1, calculations were performed assuming that a1 is always a constant value, but in the present invention, in response to changes in the value of a1, the value of a1 in the above general formula is obtained for the compound to be separated based on the TLC results at a specific solvent mixing ratio of the compound, thereby enabling the prediction of chromatography results with higher accuracy.

[0024] Furthermore, log k' = a² log B + b² (2) (however, Retention ratio:k'=(t R -t0) / t0(3) Solvent ratio: B A similar prediction can be made for a2 in the relation. In this case as well, by analyzing the results of many TLCs using a similar method, it is possible to predict the chromatographic results with higher accuracy.

[0025] An example of a method for calculating a1 and a2 is described in detail below. However, the method for calculating a1 and a2 in this invention is not limited to the method exemplified below. The cases using equation (1) and equation (2) will be described in detail below.

[0026] In this specification, B represents the solvent mixing ratio. For example, when performing liquid chromatography with a mixture of two solvents, X and Y, B represents the proportion of one of the solvents. This mixing ratio shows a certain correlation regardless of whether it is expressed as mole fraction (mol%), volume ratio (vol%), or weight ratio (wt%), so any of these can be used. In the following examples, experimental results based on mole fraction (mol%) are shown.

[0027] (When using the approximation by equation (1)) First, data is obtained by measuring the Rf values ​​of various compounds in a specific mixed solvent at various solvent mixing ratios. In this case, Rf values ​​can be obtained for various B values ​​for the same compound. These measured data are approximated as a linear equation. That is, for each compound, the measured TLC values ​​are approximated. Rf = a1B + b1 Create a relational expression. Then, these measurements and formula creation processes are performed for a large number of compounds. Figure 1 shows the obtained formulas graphed with B on the horizontal axis and Rf on the vertical axis.

[0028] As is clear from Figure 1, the slope a1 of the above general formula is clearly constant to some extent depending on the position on the two-dimensional coordinate system represented by the solvent mixing ratio B and Rf. Furthermore, if we can provide a calculation formula to clarify this relationship, we can obtain a general formula that is closer to the actual value simply by inputting the solvent mixing ratio B and Rf. Performing column chromatography using this formula is preferable because it results in a high correlation between the predicted separation and the actual separation.

[0029] Furthermore, it is also possible to create and save all the calculation formulas corresponding to each position on the Rf / B coordinate system, and then retrieve the necessary formulas by calling them up.

[0030] There are no particular limitations to the method for calculating a1 from the results shown in Figure 1, but one example is described in detail below.

[0031] As is clear from Figure 1, the slope 'a' is relatively large in the left region of the graph (i.e., the region where Rf is large even if B is small) and relatively small in the right region of the graph (i.e., the region where Rf is small even if B is large).

[0032] Therefore, for example, the relationship between the slope a1 and B can be expressed for each value when Rf is fixed. This is shown in Figure 2. As is clear from Figure 2, the relationship between the slope a1 and B becomes clear for a specific Rf value.

[0033] In other words, from the results in Figure 2, a1 = d × lnB + e (4) This relationship can be derived.

[0034] Furthermore, as can be seen from Figure 2, d in the general equation (4) above is constant for all Rf. On the other hand, the change caused by the difference in Rf is e. The relationship between Rf and the intercept e is shown in Figure 3. From Figure 3, it can be shown that there is a proportional relationship between Rf and e, and regarding this relationship, e = g × Rf + h It can be expressed as follows. Therefore, based on this relationship, we can express general equation (4) a1 = d × lnB + g × Rf + h (4 - 1) It can also be rewritten as follows. Since d, g, and h are constants determined by the mixed solvent species, a1 can be calculated by measuring B and Rf.

[0035] By using the general formula (4) created in this way, it is possible to calculate a1 for B when a particular Rf value is given. Furthermore, by preparing such formulas for many types of Rf values, a1 can be calculated for any combination of Rf and B, or a1 can be calculated from the measured values ​​of Rf and B using the general formula (4-1) above.

[0036] Based on measured values ​​for various compounds, a calculation formula for calculating a1, such as the general formula (4)(4-1) described above, can be created in advance, and a1 can be calculated by performing the calculation as described above using the calculation formula acquisition means of the preparative chromatography support apparatus of the present invention.

[0037] For example, TLC can be performed on a compound to be separated for a specific solvent mixing ratio B, the Rf value can be measured, and a1 can be calculated using the above general formula (4)(4-1) based on the relationship between B and Rf. Furthermore, the above general formula (1) can also be created. The preparative chromatography support device of the present invention has a means for obtaining a calculation formula to create such a general formula (1).

[0038] By performing this process, a general formula (1) that is closer to the results of actual column chromatography than conventional methods can be obtained for any point on the coordinate axes. Furthermore, this process does not require performing multiple TLC operations.

[0039] Alternatively, the calculation formula may be obtained by creating the above formula (1) for any point on the coordinate axis using the calculation formula described above or by other methods, storing all of these in the calculation formula acquisition means, and calling up the calculation formula (1) corresponding to the input Rf value and B.

[0040] When performing preparative column chromatography on a sample containing multiple compounds, the above-described formula acquisition means can be used to create separate formulas (1) for each of the multiple compounds to be separated. Based on the multiple formulas (1) thus obtained, the results of the column chromatography, as detailed below, can be predicted.

[0041] (When using the approximation by equation (2)) When performing an approximation based on equation (2), the examination can essentially be carried out using the same method as described above for equation (1). This approximation is fundamentally identical to that disclosed in Patent Document 2. In this invention, instead of making an approximation based on the assumption that the slope a2 is always constant when predicting chromatographic results based on equation (2), the value of a2 is calculated based on the TLC results.

[0042] Therefore, perform the same mathematical processing as that performed for the above general formula (1), and by the same method, obtain a calculation formula for calculating the a2 value corresponding to a specific Rf value, and thereby create a general formula (2) closer to the actual situation.

[0043] In the above aspect of the present invention, the following formula (3) k’=(t R -t0) / t0(3) defines the holding force k’. Here, t0 is the time required for the elution solvent to pass through a specific column, and is a column-specific value determined by the size, shape, etc. of the column. t R is the elution time required for the target sample to pass through a specific column. Since t0 is a column-specific constant, by obtaining this k’, the elution time t R when using a specific column can be obtained.

[0044] The holding force k’ has the following relationship with the solvent ratio B: logk’=alogB+b (2) [[ID=二十六]]is represented by the relationship.

[0045] To perform analysis based on such a general formula (2), it is necessary to clarify the relationship between Rf, which is the result of TLC, and the above k’. This will be described in detail below. Generally, there is a relationship between the Rf of thin-layer chromatography and t0 and t R in liquid chromatography: t R =t0 / Rf is known to hold. Therefore, if Rf is measured, the relationship between t0 and t R will be clarified, and by substituting this into the general formula (2), the k’ of the above general formula can be obtained.

[0046] Specifically, k’=(1 / Rf)-1 is. Using the formula in question, and performing the same processing as described above for formula (1), a relationship between a2 and B is created for each k'. These are then stored as means of obtaining calculation formulas. Then, except for the difference in the approximation formula, the appropriate formula (2) can be obtained based on the TLC results using the same method as formula (1) described above.

[0047] (Correction for differences between TLC and liquid chromatography) The general formulas (1) and (2) obtained by the aforementioned TLC may not perfectly match the relational formulas used in liquid chromatography.

[0048] Therefore, the general formulas (1) and (2) described above may be modified by adding a correction term to compensate for such discrepancies. Such a correction term is not specifically limited and can be modified by obtaining separation data from TLC and column chromatography, analyzing these data, and then modifying the general formulas (1) and (2).

[0049] Furthermore, when preserving the general formulas (1) and (2) for any combination of B and Rf as described above, it is also acceptable to create and preserve calculation formulas that take into account the aforementioned discrepancies within these formulas.

[0050] (Regarding the solvent species in a mixed solvent) In preparative column chromatography, when preparative chromatography is performed using a mixed solvent containing multiple types of solvents, several combinations of solvent types are possible. The relationship between B and Rf as described above is specific to the solvent type used, and if the solvent type changes, the formulas used to calculate general formulas (1) and (2) will also change.

[0051] Therefore, in the preparative chromatography support apparatus of the present invention, the means for acquiring the calculation formulas a1 and a2 as described above may store multiple types of data for multiple solvent combinations. In this case as well, for each solvent combination, an analysis based on the TLC results as described above can be performed to create a general formula, which can then be stored in the calculation formula acquisition means. In this way, the types of operations that can be supported by the preparative chromatography support device of the present invention are greatly increased, thereby enhancing its usefulness.

[0052] The combination of solvent species is not particularly limited, but a combination of a nonpolar solvent and a polar solvent is preferred. More preferably, for example, hexane / ethyl acetate, hexane / chloroform, chloroform / methanol, ethyl acetate / methanol, hexane / dichloromethane, dichloromethane / methanol, etc., can be used. Depending on the properties of the compound to be separated, an appropriate combination of solvent species can be selected from these and used.

[0053] (About gradients) A gradient pattern refers to the pattern of change that occurs when the solvent mixing ratio is changed over time during liquid chromatography. For example, it refers to the pattern shown in Figure 4. Performing a gradient itself is a common method in liquid chromatography.

[0054] If the relationship between the general formulas (1) or (2) described above is clear, the elution status when a gradient is performed by known methods, such as those described in Patent Documents 2 and 3, can be predicted. This allows for the prediction of the elution curve when preparative column chromatography is actually performed.

[0055] This allows for the calculation of the sample elution time based on the eluent mixing ratio or gradient pattern of the eluent mixing ratio, enabling a prior determination of whether separation by liquid chromatography can be achieved. Therefore, based on these calculation results, it becomes possible to select the optimal eluent mixing ratio or gradient pattern of the eluent mixing ratio that ensures sufficient separation.

[0056] (Regarding the structure of the present invention) The present invention is a preparative chromatography support device that provides useful calculation formulas for performing preparative chromatography through the calculation formula acquisition means described above. When actually applying such a preparative chromatography support device to liquid chromatography, it is preferable that the device take the form of a control device for the liquid chromatograph, a method for executing liquid chromatography, and a control program for the liquid chromatograph.

[0057] The preparative chromatography support device of the present invention is preferably a computer. Preferably, it is a computer that stores the calculation formulas and programs for performing the above-described processing. Furthermore, it may also be possible to store the program for performing such processing on a server using a network, and have a client access this program to perform the processing. The following details each of these methods.

[0058] (Measured value storage means) The above-mentioned measured value storage means is a means for storing the results of thin-layer chromatography.

[0059] Figure 5 shows a TLC apparatus 1 used for thin-layer chromatography (TLC). The TLC apparatus 1 comprises a silica gel thin-layer plate 2 on which a sample 3 is dripped, and an eluent 4 stored in a container 5. The sample 3 and the silica gel forming the silica gel thin-layer plate 2 are the same as those used in liquid chromatography, which will be described later.

[0060] Furthermore, the eluent 4 used is a solvent system similar to that used in liquid chromatography, and a mixed solvent is used.

[0061] In TLC, first, as shown in Figure 5, a thin layer of silica gel 2 on which the sample 3 is suspended is immersed in the eluent 4 as shown in Figure 1. Then, due to capillary action, the mixed solution 4 is drawn up into the silica gel layer 2. Along with this, the sample 3 also moves upward. Once the movement of the eluent 4 and the sample 3 is complete, as shown in Figure 2(b), the sample 3 moves to the position of sample 3'. At this time, the distance from the position of sample 3 before immersion in the mixed solution 4 to the upper end of the eluent 4 is taken as 1.0, and the distance to sample 3' relative to 1.0 is calculated as the mobility Rf (Figure 6).

[0062] In this case, the operator may read the Rf value from the thin-layer plate after measurement and input the value as a numerical value, or the thin-layer ctomagraphy plate may be placed in a predetermined position on the device, and the Rf value may be automatically read based on image analysis. In spot analysis, if necessary, a method may be adopted in which detection light such as ultraviolet light is applied and the resulting emission is used to read the spot position.

[0063] The measured value storage means is a means for storing the Rf value obtained by the thin-layer chromatography described above, in association with the solvent mixing ratio B. The values ​​stored in this way are used in the following calculation formula acquisition means.

[0064] (Calculation formula acquisition means) The means for obtaining the calculation formula in the present invention is a means for creating a general formula (1) for a specific solvent system for a specific component based on the results of the thin-layer chromatography described above. This calculation formula has already been described in detail.

[0065] (Method for predicting liquid chromatography results) Once general formula (1) or general formula (2) is determined for all samples requiring separation, it becomes possible to predict the eluent mixing ratio and the corresponding elution time for the column used. Based on this, the operator can easily estimate a good solvent mixing ratio.

[0066] Furthermore, by inputting specific liquid chromatography conditions (e.g., sample volume), the system can display the corresponding elution time. It can also display the resolution Rs and the expected elution curve. By viewing this information, operators can easily judge the quality of multiple liquid chromatography conditions. This allows for easy selection of the optimal liquid chromatography conditions (solvent mixing ratio, column used, etc.) before the experiment.

[0067] The creation of such liquid chromatography result predictions can be done using a standard computer, and the results can be displayed on a general display device such as various types of displays.

[0068] The elution time for such liquid chromatography can be calculated according to the methods described in the prior art, such as those in Patent Documents 2 to 4. Combining the method of the present invention with the methods described in these patent documents allows for more accurate predictions and is therefore preferable.

[0069] The presentation of liquid chromatography result predictions may include showing specific numerical values ​​such as elution time and resolution in an image, or displaying an elution curve. By comparing these values ​​under several measurement conditions, operators can determine the optimal liquid chromatography method.

[0070] Furthermore, column selection is an important factor in determining chromatography conditions. As mentioned above, sample load is also an important factor in column selection. In other words, a larger sample volume requires a larger column. Information necessary for this purpose may be shown in the image.

[0071] (Method for determining chromatography conditions) In this way, the present invention allows the operator to evaluate liquid chromatography conditions they have created or those recommended by the device, using a liquid chromatography result prediction means, and ultimately determine the liquid chromatography conditions.

[0072] (Mixing ratio control means) The present invention may also involve performing liquid chromatography based on the determination made by the liquid chromatography condition determination means described above. The mixing ratio control means is a means for outputting a control signal that controls the mixing ratio of the eluent delivered to the column or the gradient pattern of the mixing ratio of the eluent, based on the liquid chromatography conditions selected by the operator based on the liquid chromatography condition determination means. Known mixing ratio control means can be used.

[0073] (Control device for liquid chromatograph) An example of an embodiment of the liquid chromatograph control device of the present invention will be described in more detail below with reference to the figures. However, the present invention is not limited to what is shown in the following drawings. The present invention may also relate to a control device that controls liquid chromatography by the means described above, using a liquid chromatography apparatus equipped with the necessary elements and a computer that controls the apparatus. An example of how the above-described method can be implemented in an actual device is described in detail below.

[0074] Furthermore, in this invention, commonly used liquid chromatography gradient conditions may be stored as a library, and based on the results of general formulas (1) and (2) for each component, the computer may automatically select a recommended eluent mixing ratio or gradient pattern of eluent mixing ratio from the library and recommend it to the operator. The operator may determine the liquid chromatography conditions by approving the recommended conditions.

[0075] Furthermore, column selection is also an important factor in determining the chromatography conditions. For this reason, it is preferable that the t0 values ​​corresponding to each column used in the liquid chromatography are stored in the liquid chromatography result prediction means, and that the corresponding t0 value is retrieved when the operator selects a column. In addition, a function to manually input t0 may be provided as needed.

[0076] In this way, the present invention allows the operator to evaluate liquid chromatography conditions they have created or those recommended by the device, using a liquid chromatography result prediction means, and ultimately determine the liquid chromatography conditions.

[0077] (Mixing ratio control means) The present invention provides a method for performing liquid chromatography based on the determination made by the liquid chromatography condition determination means described above. The mixing ratio control means is a means for outputting a control signal that controls the mixing ratio of the eluent delivered to the column or the gradient pattern of the mixing ratio of the eluent, based on the liquid chromatography conditions selected by the operator based on the liquid chromatography condition determination means. The mixing ratio control means can be a known one, for example, one disclosed in Patent Document 1.

[0078] (Control device for liquid chromatograph) An example of an embodiment of the liquid chromatograph control device of the present invention will be described in more detail below with reference to the figures. However, the present invention is not limited to what is shown in the following drawings. The present invention relates to a control device that controls liquid chromatography by the means described above, using a liquid chromatography apparatus equipped with the necessary elements and a computer that controls the apparatus. An example of how the above-described method can be implemented in an actual device is described in detail below.

[0079] Figure 6 shows the liquid chromatograph apparatus 11. The liquid chromatograph apparatus 11 consists of a container 12 holding solvent A, a container 13 holding solvent B, a solenoid valve 14 located where solvent A and solvent B are connected, a mixer 15 holding eluent 4, a pump 16, an injector 17, a column 18, a detector 19, and a fraction collector 20, all arranged in this order to form a pathway. The liquid chromatograph control device 21 is connected to the solenoid valve 14.

[0080] Container 12 contains solvent A, and container 13 contains solvent B. Note that the number of solvents used is not limited to two; it may be increased depending on the usage conditions and purpose. Generally, solvents A and B are used in combination of nonpolar and polar molecules.

[0081] Pump 16 pumps solvent A and solvent B through container 15 and solenoid valve 14 in the circuit of the liquid chromatograph apparatus 11. The solenoid valve 14 selects which solvent to pump from solvent A or solvent B based on a control signal from the liquid chromatograph control device 21. The mixing ratio of solvents A and B in the mixer 15 is determined according to the selection time of each solvent at the solenoid valve 14. The pumped solvents A and B are temporarily stored in the mixer 15 and become the eluent 10. This eluent 10 has the calculated mixing ratio, as will be described later.

[0082] The injector 17 holds the sample 3, and the sample 3 is dispensed as the eluent 10 passes through it. Furthermore, the injector 17 is not limited to a single unit; multiple injectors can be arranged side-by-side to selectively choose a path, allowing for continuous processing of multiple samples.

[0083] Column 18 is packed with a stationary phase, and liquid chromatography is performed by passing the mixed solvent 10 through it. The stationary phase used is silica gel, which forms the silica gel thin-layer plate 2 shown in Figure 1. Note that the column is not limited to a single column; multiple columns are arranged side-by-side to selectively choose pathways, allowing for the performance of multiple types of liquid chromatography.

[0084] The detector 19 detects the results of liquid chromatography performed in column 18. The fraction collector 20 has multiple test tubes, and based on the analysis results from the detector 19, each component of the sample 3 is separated into a separate test tube.

[0085] The liquid chromatography control device of the present invention is a device for controlling liquid chromatography as shown in Figure 6, and is preferably a computer having the above-described measured value storage means, calculation formula acquisition means, liquid chromatography result prediction means, liquid chromatography condition determination means, and mixing ratio control means on an internal hard disk, or a client computer connected to a server where the information necessary to perform these means is stored.

[0086] This allows for the execution of the calculations described above, and by controlling the liquid chromatography process, good liquid chromatography results can be achieved. [Examples]

[0087] The present invention will be described in more detail below with reference to examples.

[0088] (Regarding the method for calculating a1 using TLC measurement) Multiple mixed solvents containing B% by weight of X and Y were prepared, and then TLC was performed on 12 compounds in multiple mixed solvents with different proportions of B. Rf = a1B + b1(1) The relationship was clarified. Figure 1 shows the general formula (1) for each of the 12 compounds calculated in this way, displayed on a graph.

[0089] Furthermore, based on Figure 1 and the general formula, a1 was read for each case where Rf = 0.1, 0.2, 0.3, 0.35, 0.4, 0.5, and 0.6, and the relationship between each of the resulting a1 and LogB is shown in Figure 2. From Figure 2, a1 and logB can be approximated by a linear relationship, and a relational expression showing the relationship between a1 and logB can be obtained for each Rf value.

[0090] For each Rf value obtained in this way, a relational expression showing the relationship between a1 and logB was created using the method described above and saved on a computer, so that when Rf and B are entered, the corresponding general formula (1) can be called up.

[0091] We then conducted preparative chromatography experiments using the preparative chromatography support system created in this manner.

[0092] The experiment used a test sample containing a mixture of three compounds. A single-point TLC measurement was performed using a specific mixed solvent B. Based on these results, a general formula (1) was created for each of the three compounds, and based on this, elution curves were predicted for several recommended gradient patterns. The optimal result was selected, and preparative chromatography was performed accordingly. The apparatus shown in Figure 7 was used for preparative chromatography. Furthermore, detection was performed simultaneously with preparative chromatography, and the elution curve was created during the preparative process.

[0093] The results are shown in Figure 8. In Figure 8, the horizontal axis represents time (minutes) and the vertical axis represents peak intensity. Furthermore, Figure 8 shows the predicted elution time of the final compound when prediction was made using relational equation (1) obtained by the preparative chromatography support device of the present invention. From these results, it is clear that the target compound was eluted at an elution time close to the prediction.

[0094] As described in Patent Document 1, a similar experiment was conducted based on the TLC results using a fixed a1 value. The results are shown in Figure 9.

[0095] Comparing these results, there is a difference in the predicted elution time. Furthermore, when actual preparative column chromatography was performed, the predicted results based on the preparative chromatography support device of the present invention were closer to the actual preparative column chromatography results. From this, it can be concluded that the preparative chromatography support device of the present invention has superior performance compared to conventional similar devices. [Industrial applicability]

[0096] The preparative chromatography support device of the present invention can be suitably used in preparative chromatography in chemical research. [Explanation of symbols]

[0097] 1 TLC device 2. Thin silica gel plate 3 Samples 4. Eluent 5 containers 11. Liquid Chromatography Apparatus 12, 13 containers 14 Solenoid valve 15 Mixer 16 pumps 17 Injectors 18 columns 20 Fraction Collectors

Claims

1. A preparative chromatography support device equipped with a means for obtaining calculation formulas, This device provides information regarding preparative chromatography in column chromatography based on the measured values ​​B and Rf, which are the actual values ​​obtained from a single measurement using a mixed solvent of two specific solvents at a mixing ratio B, as measured by thin-layer chromatography (TLC). The means of obtaining the calculation formula is, Rf=a １ B+b １ (1) a in the relational expression １ or logk’=a ２ logB+b ２ (2) (However, retention ratio: k' = (t Ｒ -t ０ ) / t ０ (3) Solvent ratio: B a in the relational expression ２ This is obtained based on the measured values ​​B and Rf, which are the actual results of a single measurement in TLC. A preparative chromatography support apparatus characterized by having a means for obtaining a calculation formula based on data obtained by actually measuring the relationship between the Rf value and the solvent mixing ratio B for a large number of compounds for a specific combination of two solvents.

2. A preparative chromatography apparatus characterized by comprising the preparative chromatography support apparatus described in claim 1.

3. (1) A step in which thin-layer chromatography of the compound to be separated is performed in a mixed solvent of two specific solvents at a mixing ratio B. Step (2) inputs the Rf value and mixing ratio B obtained in step (1) into the preparative chromatography support device described in claim 1. Based on the data input in step (2), the preparative chromatography support system calculates Rf = a １ B + b (1) or logk’=a ２ logB+b (2) (however, Keep ratio: k' = (t) Ｒ -t ０ ) / t ０ (3) a in the relational expression １ or a ２ Step (3) to obtain Step (4) to determine the gradient conditions in preparative chromatography based on the said relational expression. and Step (5) is to perform chromatography according to the gradient conditions determined in step (4). A preparative chromatography method characterized by having [a certain feature].

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