JP4885060B2 - Reverse phase liquid chromatograph control apparatus, reverse phase liquid chromatography execution method, and reverse phase liquid chromatograph control program - Google Patents

Description

  The present invention relates to reverse phase liquid chromatography, and more particularly, to examination of conditions when performing reverse phase liquid chromatography.

  Conventionally, liquid chromatography that separates each component in a sample by passing a sample dissolved in a mobile phase, which is an eluent mixed with a plurality of solvents, through a stationary phase packed in a column is known. ing. In such chromatography, each component in the sample introduced into the column is discharged from the column because the time for passing through the column differs depending on the affinity with the eluent and the interaction with the stationary phase of the column. At this point, each component is separated.

Separation degree R _S in liquid chromatography as described above, the sample increases monotonically with to increase the elution time T _r to elute saturates the elution time T _r is sufficiently large to a predetermined size . That is, when the elution time _Tr is short, the components cannot be sufficiently separated. On the other hand, when the elution time _Tr is excessively long, the time and the eluent are wasted. Here, the elution time _Tr changes depending on the mixing ratio condition of the eluent. Therefore, when performing liquid chromatography, the elution time _Tr should be such that the elution time _Tr is long enough to ensure that the time required for the separation of the components is sufficient while not wasting time and eluent waste. It is necessary to adjust the mixing ratio condition.

Patent Document 1 describes, as a preliminary experiment, a method of adjusting a mixing ratio condition of an eluent by performing thin layer chromatography (TLC) using a substance used as a stationary phase in liquid chromatography as a thin layer. Thin-layer chromatography is performed by immersing a thin-layered stationary phase in an eluent serving as a mobile phase, and the mobility of the sample relative to the eluent by the action of the eluent being sucked into the thin layer by capillary action and the sample being sucked up. R _f can be determined. Here, when the eluent has a specific mixing ratio, the elution index T ₀ / T _r (T ₀ : the time from when the eluent starts to flow into the gap of the column until it starts to flow) measured by liquid chromatography, The mobility _Rf measured by thin layer chromatography has a correlation that they are equal to each other. Further, the relationship between the eluent mixture ratio and the mobility _Rf is a proportional relationship, and the rate of change of the mobility _Rf relative to the change in the eluent mixture ratio is constant even when the type of the sample is changed.

That is, as a preliminary experiment, thin layer chromatography is first performed using an eluent having a specific mixing ratio, and the mobility _Rf is measured. At this time, due to the property that the change rate of the mobility _Rf with respect to the change in the mixing ratio of the eluent is constant, a straight line of the graph with the horizontal axis indicating the mixing ratio and the vertical axis indicating the mobility _Rf is uniquely determined. Furthermore, when considering the relationship that the mobility R _f is equal to the elution index T ₀ / T _r in liquid chromatography, the mixing ratio of the eluent that gives the desired elution index T ₀ / T _r can be determined from this graph.
JP 2003-240765 A

  Here, in reverse phase liquid chromatography in which separation is performed by hydrophobic interaction between a stationary phase and a sample, octadecylsilylated silica gel (ODS) in which octadecyl (C18) group is chemically bonded to the surface of the silica gel is generally used as the stationary phase. Is used. Since ODS has high water repellency, when thin layer chromatography is performed using ODS as a thin layer, it is difficult for the eluent or sample to enter the thin layer. Therefore, when performing reverse-phase liquid chromatography, it is difficult to accurately perform thin-layer chromatography, which is a preliminary experiment, and the method for adjusting the mixing ratio condition of the eluent described in Patent Document 1 is applied. I can't.

Therefore, at present, in order to adjust the mixing ratio conditions of the eluent when performing reversed-phase liquid chromatography, first, preliminary experiments are repeatedly performed while changing the mixing ratio of the eluent, and a mixture that provides a suitable elution time _Tr is obtained. Search for the ratio. Therefore, the work is complicated and time consuming, and there is a problem that a large amount of eluent is required.

Therefore, an object of the present invention is to provide a reverse phase liquid chromatography control device capable of executing reverse phase liquid chromatography under an eluent condition in which the elution time _Tr is appropriate without performing complicated work, An execution method and a control program for reversed-phase liquid chromatography are provided.

The control apparatus for the reverse phase liquid chromatograph of the present invention uses a larger amount of sample than the sample used in the preliminary reverse phase liquid chromatography performed in advance, and the organic solvent contained in the eluent composed of a plurality of components. This is a control apparatus for a reverse phase liquid chromatograph that performs control so that a reverse phase liquid chromatograph having a concentration at which the specific elution index T ₀ / T _r0 obtained by the preliminary reverse phase liquid chromatography is obtained is performed. Te, a first density control means for outputting a control signal for controlling the concentration of the organic solvent contained in the eluent in performing reverse phase liquid chromatography of the preliminary using the eluent, reverse phase of the preliminary a plurality of components to be measured in the liquid chromatography, the eluent is included in the time T ₀ and the sample to begin to flow out from the start to flow into the void of the column Elution index T ₀ / T _r a particular component determined by the elution time T _r until elution of out, the elution time integral value of the concentration of the organic solvent in until the elution time T _r in the chromatographic Actual value storage means for storing in association with a specific concentration of the organic solvent, which is a value divided by the time T _r , and a rate of change of the elution index T ₀ / T _r with respect to a change in the concentration of the organic solvent in the eluent a change rate storage means for storing, and elution index T ₀ / T _r with respect to the particular concentration stored in the measured value storing means, the elution index T ₀ / T _r stored in the change rate storage unit Based on the change rate, the concentration calculating means for obtaining the concentration of the organic solvent of the eluent that obtains the specific elution index T ₀ / T _r0, and based on the calculation result of the concentration calculating means, elution index T ₀ / T _r And a second density control means for outputting a control signal for controlling the concentration of the organic solvent in the eluting solution to be fed to the column so that the specific dissolution index T ₀ / T _r0.

In addition, the method for performing reverse phase liquid chromatography of the present invention uses a larger amount of sample than the sample used in preliminary reverse phase liquid chromatography performed in advance, and an organic solvent contained in an eluent composed of a plurality of components. It is the density, a run method of a reverse-phase liquid chromatography as a specific concentration elution index T ₀ / T _r0 is obtained which is determined by reverse-phase liquid chromatography of the preliminary, opposite the preliminary using the eluent By performing phase liquid chromatography, the time T ₀ from when the eluent starts to flow into the column gap until it begins to flow and the elution time until the specific component of the plurality of components contained in the sample elutes a first step of obtaining an elution index T ₀ / T _r determined by T _r, the first elution obtained in step index T ₀ / T _r, and reverse-phase liquid in the pre-b On the basis of the specific concentration of the organic solvent which is the value obtained by dividing by the elution time T _r the time integral value of the concentration of the organic solvent contained in the eluent in until elution time T _r in Togurafi, the elution By using the rate of change of the elution index T ₀ / T _r of the sample with respect to the change in the concentration of the organic solvent in the liquid, the concentration of the organic solvent in the eluent that obtains the specific elution index T ₀ / T _r0 is obtained. A second step, and a third step of feeding the eluent having a concentration of the organic solvent that provides the specific elution index T ₀ / T _r0 obtained in the second step to a column. It has.

Furthermore, the control program for reversed-phase liquid chromatography of the present invention uses a larger amount of sample than the sample used in the preliminary reversed-phase liquid chromatography performed in advance, and the organic solvent contained in the eluent composed of a plurality of components. Reverse phase liquid chromatograph for causing a computer to function so that a reverse phase liquid chromatograph is obtained at a concentration at which the specific elution index T ₀ / T _r0 obtained by the preliminary reverse phase liquid chromatography is obtained a control program, a first density control means for outputting a control signal for controlling the concentration of the organic solvent contained in the eluent in performing reverse phase liquid chromatography of the preliminary using the eluent, the Measured in preliminary reverse phase liquid chromatography until eluent begins to flow into the column gap and then begins to flow out Elution index T ₀ / T _r a particular component determined by the elution time T _r until elution of a plurality of components included in the time T ₀ and the sample, up to the elution time T _r in the chromatographic Measured value storage means for storing in association with a specific concentration of the organic solvent, which is a value obtained by dividing the time integral value of the concentration of the organic solvent by the elution time _Tr , and the concentration of the organic solvent in the eluent the change rate storage means for storing the rate of change in dissolution index T ₀ / T _r relative to the change, and eluted index T ₀ / T _r with respect to the particular concentration stored in the measured value storing means, the change rate storage unit Concentration calculating means for determining the concentration of the organic solvent in the eluent that gives the specific elution index T ₀ / T _r0 based on the rate of change of the elution index T ₀ / T _r stored in Performance with concentration calculation means Based on the results, a control signal for controlling the concentration of the organic solvent in the eluent eluted index T ₀ / T _r is fed to the column so that the specific dissolution index T ₀ / T _r0 sample output The computer is caused to function as second density control means for the purpose.

  The above-mentioned program can be recorded and distributed on a removable recording medium such as a CD-ROM, FD, or MO, or a fixed recording medium such as a hard disk, or can be communicated over the Internet by wired or wireless telecommunication means. Distribution is possible via a network.

According to these configurations, if the elution index T ₀ / T _r of the sample at an arbitrary specific concentration is obtained by preliminary reverse phase liquid chromatography, the concentration of the eluent at the desired specific elution index T ₀ / T _r0 can be determined. I can know. Therefore, reverse-phase liquid chromatography can be performed under the eluent conditions that provide an appropriate elution index T ₀ / T _r0 without performing complicated operations.

  In the control device for a reverse phase liquid chromatograph of the present invention, the first concentration control means may output a control signal for keeping the concentration of the organic solvent in the eluent constant.

  In the method for performing reverse phase liquid chromatography according to the present invention, the preliminary reverse phase liquid chromatography may be performed with a constant concentration of the organic solvent in the eluent.

  According to these configurations, preliminary reverse phase liquid chromatography can be easily performed.

  In the control device for a reverse phase liquid chromatograph of the present invention, the first concentration control means may output a control signal for changing the concentration of the organic solvent in the eluent with time.

  In the method for performing reverse phase liquid chromatography according to the present invention, the preliminary reverse phase liquid chromatography may be performed while changing the concentration of the organic solvent in the eluent over time.

  According to these configurations, it is possible to avoid a very long time until the sample is eluted, as in the case of preliminary reverse phase liquid chromatography with a constant organic solvent concentration in the eluent. Can do.

In the control apparatus for a reverse phase liquid chromatograph according to the present invention, the change rate storage means is configured so that the elution index T ₀ / T of the sample with respect to the change in the concentration of the organic solvent for each different type of eluent having a different combination of components It is preferable that the rate of change of _r can be stored.

  According to this configuration, the controller of the reverse phase liquid chromatograph can be used for various types of eluents having different combinations of the components.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.
<First Embodiment>
FIG. 1 is a diagram showing a schematic configuration of a reverse phase liquid chromatograph apparatus according to the present embodiment. As shown in FIG. 1, the reverse phase liquid chromatograph 11 is provided at a position where the container 12 storing the solvent A, the container 13 storing the solvent B, and the solvent A and the solvent B are connected. The electromagnetic valve 14, the mixer 15 in which the eluent 10 is stored, the pump 16, the injector 17, the column 18, the detector 19, and the fraction collector 20 are arranged in this order to form a path. Yes. A reverse phase liquid chromatograph control device 21 is connected to the electromagnetic valve 14.

  The container 12 stores the solvent A, and the container 13 stores the solvent B. In addition, the solvent used is not limited to two types, You may increase a number according to a use condition and the objective. Generally, the solvent A and the solvent B are used in combination with a polar solvent and an organic solvent.

  The pump 16 pumps up the solvent A and the solvent B through the mixer 15 and the electromagnetic valve 14 in the circuit of the reverse phase liquid chromatograph apparatus 11. The solenoid valve 14 selects the solvent to be pumped up from the solvent A or the solvent B by the control signal from the reverse phase liquid chromatograph control device 21. The mixing ratio of the solvents A and B in the mixer 15 is determined according to the selection time of each solvent in the electromagnetic valve 14. In the mixer 15, the solvent A and the solvent B that have been pumped up are temporarily stored and used as the eluent 10. The eluent 10 has a mixing ratio in which the concentration of the organic solvent becomes a concentration calculated by the concentration calculator 22a (see FIG. 2) of the reverse phase liquid chromatograph controller 21 as described later.

The injector 17 has the sample 3, and the sample 3 is sent out when the eluent 10 passes. Note that the number of injectors 17 is not limited to one, and a plurality of injectors that can be selectively selected can be used, and a plurality of samples can be continuously operated.

  The column 18 is filled with a stationary phase, and reverse phase liquid chromatography is performed by passing the eluent 10. In the present embodiment, ODS is used as the stationary phase. Note that the number of columns is not limited to one, and it is possible to perform a plurality of types of reversed-phase liquid chromatography using a plurality of columns arranged side by side so that a route can be selectively selected.

  The detector 19 detects the result of the reverse phase liquid chromatography performed in the column 18. The fraction collector 20 has a plurality of test tubes, and is separated into each test tube for each component contained in the sample 3 based on the analysis result of the detector 19.

  Moreover, the reverse phase liquid chromatograph control device 21 is configured by an information processing device such as a general-purpose personal computer, for example, and as shown in FIG. 2, the concentration calculation unit 22a, the first concentration control unit 22b, and the first 2, a CPU 22 having a density control unit 22 c, a memory 25 having a measured value storage unit 25 a and a change rate storage unit 25 b, a hard disk 26, and an input / output unit 28. The input / output unit 28 includes, for example, an input unit 29 configured by a keyboard, a mouse, and the like, a display 30 for notifying an operator of various information in the execution process of reversed-phase liquid chromatography, and the first and second of the CPU 22. An electromagnetic valve operating unit 31 that sends an open / close signal of the electromagnetic valve 14 based on an instruction from the concentration control units 22b and 22c is connected.

  The hard disk 26 stores various software including the control program for the reverse phase liquid chromatograph according to the present embodiment. The above-described units 22a, 22b, 22c, 25a, and 25b are constructed by combining these hardware and software. This program can be installed in any computer by recording it on a removable recording medium such as a CD-ROM, FD, or MO.

The actual measurement value storage unit 25a uses the reverse-phase liquid chromatograph apparatus 11 to start elution index measurement by a preliminary reverse-phase liquid chromatography performed on a small amount of the sample 3 before starting the full-scale sorting operation of the sample 3. T ₀ / T _r is stored in relation to the specific concentration of the organic solvent in the chromatography. T ₀ is the time from when the eluent 10 starts to flow into the gap of the column 18 until it starts to flow out in reversed-phase liquid chromatography, and T _r is the elution time until the sample 3 is eluted. Here, the specific concentration in the reversed-phase liquid chromatography of spare time integral of the concentration of the organic solvent in until elution time T _r, namely the cumulative concentration of the organic solvent, be a value obtained by dividing the elution time T _r Mean the average concentration of the organic solvent up to the elution time _Tr . Data to be stored in the actual value storage unit 25a may be input from the input unit 29 by the operator.

In the present embodiment, the preliminary reverse-phase liquid chromatography is performed in an isocratic mode in which the concentration of the organic solvent contained in the eluent is constant as shown in FIG. Therefore, as shown in FIG. 3, when the elution time in the preliminary reverse phase liquid chromatography using the eluent having the organic solvent concentration of B ₁ is T _r1 , the measured value storage unit 25a includes T ₀ / T _r1 is stored as the elution index T ₀ / T _r for the specific concentration B ₁ .

The change rate storage unit 25b stores the change rate of the elution index T ₀ / _Tr of the sample 3 with respect to the change in the concentration of the organic solvent in the eluent. Here, the relationship between the concentration of the organic solvent contained in the eluent and the elution index T ₀ / T _r of the sample 3 is a proportional relationship. That is, the change rate of the elution index T ₀ / T _r, as shown in FIG. 4, the concentration of the organic solvent on the horizontal axis, when the vertical axis depicts the graph as an elution index T ₀ / T _r of the straight line slope Indicated by Note that the rate of change of the elution index T ₀ / T _r is constant even when the type of sample is changed, as indicated by a broken line in FIG. In addition, as shown by the alternate long and short dash line in FIG. 4, when different types of eluents having different combinations of components are used, different values are taken.

As shown in FIG. 5, the rate of change of the elution index T ₀ / _Tr is determined in advance for a plurality of reverse-phase liquid chromatography in isocratic mode in which the concentration of the organic solvent contained in the eluent 10 is constant. It is obtained by performing once. That is, it can be seen from FIG. 5 that the elution times T _r when reverse phase liquid chromatography is performed with the concentration of the organic solvent being 40%, 30%, 20%, 10% are aT ₀ , bT ₀ , cT ₀ , dT ₀ , respectively. It becomes. Therefore, the elution index T ₀ / _Tr when the concentration of the organic solvent is 40%, 30%, 20%, 10% is 1 / a, 1 / b, 1 / c, 1 / d, respectively. I understand. Accordingly, the rate of change of the elution index T ₀ / _Tr can be obtained by plotting a graph as shown in FIG. 4 and obtaining the slope thereof.

The hard disk 26 stores the rate of change of the elution index T ₀ / T _r for different types of eluents with different combinations of components. Then, when the control program according to the present embodiment is executed, only the rate of change of the elution index T ₀ / T _r for the type of eluent of the specific concentration stored in the measured value storage unit 25 a is loaded into the memory 25. Is done.

The concentration calculation unit 22a has an actual measurement value of the elution index T ₀ / T _r for a specific concentration stored in the actual measurement value storage unit 25a, and a change rate of the elution index T ₀ / T _r stored in the change rate storage unit 25b. Based on the above, the concentration of the organic solvent in the eluent from which the specific elution index T ₀ / T _r0 is obtained is obtained. That is, for example, when the elution index stored in the measured value storage unit 25a is T ₀ / T _r1 and the specific concentration is B ₁ , the horizontal axis represents the concentration of the organic solvent, and the vertical axis represents the elution index T. ₀ / T straight graph with _r, as shown in FIG. 6, the coordinates _{(B 1, T 0 / T} r1) through, and the change rate of the elution index T ₀ / T _r stored in the storage unit 25b It is uniquely determined in the same solvent system as a straight line having a slope according to the rate of change. Therefore, if the slope of the straight line shown in FIG. 6 is F, the concentration B _{0 of the} organic solvent that provides the desired specific elution index T ₀ / T _r0 can be obtained by the following (Equation 1).

Here, the specific elution index T ₀ / T _r0 will be described. Generally, in liquid chromatography, separation degree R becomes optimal when the elution time T _r is 4T _0. Accordingly, in this embodiment, the elution time performing reverse phase liquid chromatography T _r is the be adjusted to be 4T _0. However, the elution time _Tr does not necessarily have to be adjusted to 4T _0, and may be adjusted to a different value. For example, when full-scale reverse phase liquid chromatography is performed in the isocratic mode as shown in FIG. 7, the eluent 10 has an organic solvent concentration such that the elution index T ₀ / T _r is 0.25. Is used, the elution time T _r of the sample 3 becomes 4T ₀ . Therefore, when elution is performed in the isocratic mode, the concentration calculator 22a obtains the organic solvent concentration B ₀ such that the specific elution index T ₀ / T _r0 is 0.25.

Further, for example, as shown in FIG. 8, during the time width “W (wait)” until the elapsed time reaches 1T ₀ , the organic solvent having an elution index T ₀ / T _r of 0.2 is used. The concentration of the organic solvent contained in the eluent 10 is equal to the elution index T ₀ / T _r during the time width “G (gradient)” until the elapsed time reaches 4T ₀ after using the eluent 10 having the concentration. Is gradually changed from a concentration corresponding to 0.2 to a concentration corresponding to 0.5, and then the elution index during the time period “T (top)” until the elapsed time reaches 6T _0. When full-scale reverse phase liquid chromatography is performed in a gradient mode using an eluent 10 having an organic solvent concentration such that T ₀ / T _r is 0.5, the elution time T _{r of} sample 3 is 4T _0. It becomes. Therefore, when elution is performed in such a gradient mode, the concentration calculation unit 22a uses the organic solvent concentration B _0w and the specific elution index T such that the specific elution index T ₀ / T _r0w is 0.2. _Determine the concentration B _{0t of the} organic solvent such that ₀ / T _r0t is 0.5.

The first concentration control unit 22b outputs a control signal for controlling the concentration of the organic solvent contained in the eluent 10 fed to the column 18 when performing preliminary reverse phase liquid chromatography. As described above, in the present embodiment, preliminary reverse-phase liquid chromatography is performed in the isocratic mode. Therefore, the first concentration control unit 22b generates a control signal that always keeps the concentration of the organic solvent constant. Output. The second concentration control unit 22c is fed to the column 18 based on the calculation result in the concentration calculation unit 22a so that the elution index T ₀ / T _{r of} the sample 3 becomes the specific elution index T ₀ / T _r0. A control signal for controlling the concentration of the organic solvent contained in the eluent 10 is output. The control signals output from the first and second concentration control units 22 b and 22 c are both supplied from the input / output unit 28 to the electromagnetic valve operation unit 31.

  Next, a method for performing reverse phase liquid chromatography will be described with reference to FIG. 9 which is a flowchart showing a processing procedure in the reverse phase liquid chromatograph apparatus 11 in the present embodiment.

First, the operator repeatedly determines whether or not an instruction to perform preliminary reverse-phase liquid chromatography is input from the input unit 29 (S1). When an instruction to execute preliminary reverse phase liquid chromatography is input (S1: YES), preliminary reverse phase liquid chromatography is executed (S2). At this time, the first concentration control unit 22 b outputs a control signal to the electromagnetic valve operation unit 31 such that the eluent 10 in which the concentration of the organic solvent is kept constant is sent to the column 18. Subsequently, the actual measurement value of the elution index T ₀ / T _r of the sample 3 in the preliminary reversed-phase liquid chromatography and the specific concentration of the eluent in the chromatography are stored in the actual measurement value storage unit 25a of the memory 25 (S3).

Next, for the type of eluent stored in the measured value storage unit 25a, data on the rate of change of the elution index T ₀ / T _r of the sample 3 with respect to the change in the concentration of the organic solvent is read from the hard disk 26. Is loaded into the change rate storage unit 25b (S4).

Subsequently, the concentration calculation portion 22a, the measured value of the elution index T ₀ / T _r for a particular concentration stored in the measured value storage section 25a, the elution index T ₀ / T _r which is stored in the change rate storage unit 25b Based on the rate of change, the relationship between the concentration of the organic solvent contained in the eluent 10 and the elution index T ₀ / T _r of the sample 3 is determined one-to-one as shown in FIG. 6 (S5). Here, it is repeatedly determined whether or not the elution mode when the sample 3 is fully collected by the reverse phase liquid chromatograph 11 is input from the input unit 29 by the operator (S6).

When the elution mode is input (S6: YES), the concentration calculation unit 22a sets the concentration for the specific elution index T ₀ / T _r0 required when performing full-scale reverse phase liquid chromatography in step S5. Calculation is made based on the defined one-to-one relationship (S7). That is, in step S6, when an instruction to perform elution in the isocratic mode as shown in FIG. 7 is input, the concentration calculator 22a, as described above, specifies the specific elution index T ₀ / T _r0. The organic solvent concentration B ₀ is determined so that becomes 0.25. When an instruction to perform elution in the gradient mode as shown in FIG. 8 is input, the concentration calculating unit 22a has the specific elution index T ₀ / T _r0w of 0.2 as described above. The organic solvent concentration B _0w and the organic solvent concentration B _0t such that the specific elution index T ₀ / T _r0t is 0.5 are determined.

Then, the second concentration control unit 22c operates a solenoid valve to control a control signal so that the eluent 10 is sent to the column 18 for a predetermined time so that the concentration of the organic solvent becomes the concentration B obtained in step S7. The data are sequentially output to the unit 31 (S8). The electromagnetic valve operating unit 31 supplies a signal for opening and closing the electromagnetic valve 14 to the electromagnetic valve 14 based on this control signal. As a result, the elution index T ₀ / T _r of the sample in the column 18 can be reliably set to the specific elution index T ₀ / T _r0 or in the vicinity thereof, so that a good sample can be separated. .

  Moreover, in this Embodiment, since a preliminary | backup reverse phase liquid chromatography is performed in isocratic mode, control by the 1st concentration control part 22b is simplified.

Further, in the present embodiment, the rate of change in the elution index T ₀ / T _r of the sample 3 with respect to the change in the concentration of the organic solvent is stored in the hard disk 26 for each of different types of eluents having different combinations of the contained components. Only the necessary change rate can be stored in the change rate storage unit 25b. Therefore, the reverse phase liquid chromatograph control device 21 can be used for various types of eluents having different combinations of the components.

<Second Embodiment>
Next, a second embodiment of the present invention will be described with reference to FIG. FIG. 10 is a graph for explaining preliminary reverse phase liquid chromatography according to the present embodiment. This embodiment is substantially the same as the first embodiment described above except for the aspect of preliminary reverse phase liquid chromatography. In the following description, components having the same configurations as those of the first embodiment are denoted by the same reference numerals, and description thereof is omitted as appropriate.

In the present embodiment, the first concentration control unit 22b is included in the eluent 10 fed to the column 18 so that preliminary reverse-phase liquid chromatography is performed in a gradient mode as shown in FIG. A control signal for controlling the concentration of the organic solvent is output. That is, until the elapsed time reaches 4T ₀ , the concentration of the organic solvent contained in the eluent 10 gradually changes from 0% to 100%, and then the concentration of the organic solvent is maintained at 100%. The measured value storage unit 25a stores the elution index T ₀ / T _r measured by the preliminary reverse phase liquid chromatography performed in the gradient mode in association with the specific concentration of the organic solvent in the chromatography. To do. Here, as described above, the specific concentration is a value obtained by dividing the time integral value of the concentration of the organic solvent in the elution time T _r in until elution time T _r.

Note that the time integrated value of the concentration of the organic solvent until the elution time _Tr is an integrated concentration, and thus the area of the portion indicated by hatching in FIG. Accordingly, performed when a certain concentration B ₁ and divided by the time integral value elution time T _r, the area of the portion indicated by hatching in FIG. 11 (a), the Isokuratekku elution by time T _r in a specific concentration B ₁ In FIG. 11 (b), which is the integrated density at this time, it becomes equal to the area of the portion indicated by the oblique lines.

Similar to the first embodiment, the concentration calculation unit 22a stores the measured value of the elution index T ₀ / _Tr for the specific concentration stored in the measured value storage unit 25a and the change rate storage unit 25b. Based on the change rate of the elution index T ₀ / T _r , the concentration of the organic solvent in the eluent 10 from which the specific elution index T ₀ / T _r0 is obtained is obtained, and the second concentration control unit 22c Based on the calculation result at 22a, a control signal for controlling the concentration of the organic solvent contained in the eluent 10 fed to the column 18 is output. Therefore, as in the first embodiment, the elution index T ₀ / T _r of the sample 3 in the column 18 can be reliably set to the specific elution index T ₀ / T _r0 or in the vicinity thereof, which is favorable. The sample 3 can be separated.

  In this embodiment, since the preliminary reverse-phase liquid chromatography is performed in the gradient mode, the time until the sample 3 is eluted is extremely different from the case where the preliminary reverse-phase liquid chromatography is performed in the isocratic mode. Can be avoided.

The preferred embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments, and various design changes can be made as long as they are described in the claims. For example, the specific configuration of the reverse phase liquid chromatograph apparatus 11 can be changed to various forms. Further, the change rate storage unit 25b may be able to store the change rate of the elution index T ₀ / _Tr of the sample 3 with respect to the change in the concentration of the organic solvent for only one type of eluent. Moreover, the hardware configuration of the reverse phase liquid chromatograph control device 21 can be arbitrarily changed.

It is a figure which shows schematic structure of the reverse phase liquid chromatograph apparatus concerning the 1st Embodiment of this invention. It is a block diagram which shows schematic structure of the reverse phase liquid chromatograph control apparatus shown in FIG. It is a figure explaining the preliminary | backup reverse phase liquid chromatography performed with the reverse phase liquid chromatograph apparatus shown in FIG. It is the figure which showed the change rate of the elution index with respect to the change of the density | concentration of the organic solvent memorize | stored in the change rate memory | storage part shown in FIG. It is a figure explaining the method of calculating | requiring the change rate of the elution index | exponent with respect to the change of the density | concentration of the organic solvent memorize | stored in the change rate memory | storage part shown in FIG. It is a figure explaining the calculation method of the density | concentration in the density | concentration calculating part shown in FIG. It is an example of the elution mode at the time of performing a full-scale sample collection with the reverse phase liquid chromatograph apparatus shown in FIG. 1, and is a figure which shows isocratic mode. FIG. 2 is an example of an elution mode when a sample is collected in earnest with the reverse phase liquid chromatograph apparatus shown in FIG. 1 and is a diagram showing a gradient mode. It is a flowchart which shows the process sequence in the reverse phase liquid chromatograph apparatus shown in FIG. It is a figure explaining the preliminary | backup reverse phase liquid chromatography concerning the 2nd Embodiment of this invention. It is a figure explaining the specific density | concentration at the time of performing the preliminary | backup reverse phase liquid chromatography shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Eluent 11 Reverse phase liquid chromatograph apparatus 18 Column 21 Reverse phase liquid chromatograph control apparatus 22a Concentration calculating part (concentration calculating means)
22b 1st density | concentration control part (1st density | concentration control means)
22c 2nd density control part (2nd density control means)
25a Actual value storage unit (actual value storage means)
25b Change rate storage unit (change rate storage means)

Claims (8)

A larger amount of sample is used than the sample used in the preliminary reverse-phase liquid chromatography performed in advance, and the concentration of the organic solvent contained in the eluent composed of a plurality of components is determined by the preliminary reverse-phase liquid chromatography. A control apparatus for a reverse phase liquid chromatograph for controlling so that a reverse phase liquid chromatograph having a concentration at which a specific elution index T ₀ / T _r0 is obtained,
A first density control means for outputting a control signal for controlling the concentration of the organic solvent contained in the eluent in performing reverse phase liquid chromatography of the preliminary using the eluent,
As measured in the preliminary reversed-phase liquid chromatography, a time T ₀ from when the eluent starts to flow into the gap in the column until it starts to flow and a specific component out of a plurality of components contained in the sample is eluted. elution index T ₀ / T _r determined by the elution time T _r until, at a value of the divided time integral value of the concentration of organic solvents in the elution time T _r in until the elution time T _r in the chromatographic Measured value storage means for storing in association with a specific concentration of the organic solvent;
A change rate storage means for storing a change rate of the elution index T ₀ / T _r with respect to a change in the concentration of the organic solvent in the eluent;
Based on the elution index T ₀ / T _r for the specific concentration stored in the measured value storage means and the change rate of the elution index T ₀ / T _r stored in the change rate storage means, the specific elution index T Concentration calculating means for determining the concentration of the organic solvent in the eluent from which ₀ / T _r0 is obtained;
Based on the calculation result of the concentration calculation means, the concentration of the organic solvent in the eluent sent to the column so that the elution index T ₀ / T _{r of} the sample becomes the specific elution index T ₀ / T _r0. And a second concentration control means for outputting a control signal for controlling the reverse phase liquid chromatograph.   2. The control apparatus for a reverse phase liquid chromatograph according to claim 1, wherein the first concentration control means outputs a control signal that makes the concentration of the organic solvent in the eluent constant.   2. The control apparatus for reversed phase liquid chromatography according to claim 1, wherein the first concentration control means outputs a control signal for changing the concentration of the organic solvent in the eluent with time. The rate-of-change storage means can store the rate of change of the elution index T ₀ / T _r of the sample with respect to the change in the concentration of the organic solvent, for each of different types of eluents having different combinations of components. The control apparatus of the reverse phase liquid chromatograph of any one of Claims 1-3 characterized by these. A larger amount of sample is used than the sample used in the preliminary reverse-phase liquid chromatography performed in advance, and the concentration of the organic solvent contained in the eluent composed of a plurality of components is determined by the preliminary reverse-phase liquid chromatography. A method for performing reverse phase liquid chromatography with a concentration at which a specific elution index T ₀ / T _r0 is obtained,
By performing the reversed-phase liquid chromatography of the preliminary using the eluent, eluent of a plurality of components included in the time T ₀ and the sample to begin to flow out from the start to flow into the void of the column A first step of obtaining an elution index T ₀ / T _r determined by an elution time T _r until a specific component is eluted;
Time integration of the elution index T ₀ / T _r determined in the first step and the concentration of the organic solvent contained in the eluent up to the elution time T _{r in the} preliminary reversed-phase liquid chromatography Based on the specific concentration of the organic solvent obtained by dividing the value by the elution time T _r , the change rate of the elution index T ₀ / T _r of the sample with respect to the change in the concentration of the organic solvent in the eluent is used. A second step of determining the concentration of the organic solvent in the eluent to obtain a specific elution index T ₀ / T _r0 ,
And a third step of feeding the eluent having a concentration of the organic solvent that provides the specific elution index T ₀ / T _r0 obtained in the second step to a column. To perform reverse phase liquid chromatography.   6. The method for performing reverse phase liquid chromatography according to claim 5, wherein the preliminary reverse phase liquid chromatography is performed with a constant concentration of the organic solvent in the eluent.   6. The method for performing reverse phase liquid chromatography according to claim 5, wherein the preliminary reverse phase liquid chromatography is performed while changing the concentration of the organic solvent in the eluent over time. A larger amount of sample is used than the sample used in the preliminary reverse-phase liquid chromatography performed in advance, and the concentration of the organic solvent contained in the eluent composed of a plurality of components is determined by the preliminary reverse-phase liquid chromatography. A reverse phase liquid chromatograph control program for causing a computer to perform a reverse phase liquid chromatograph with a concentration at which a specific elution index T ₀ / T _r0 is obtained,
First density control means for outputting a control signal for controlling the concentration of the organic solvent contained in the eluent in performing reverse phase liquid chromatography of the preliminary using the eluent,
As measured in the preliminary reversed-phase liquid chromatography, a time T ₀ from when the eluent starts to flow into the gap in the column until it starts to flow and a specific component out of a plurality of components contained in the sample is eluted. elution index T ₀ / T _r determined by the elution time T _r until, at a value of the divided time integral value of the concentration of organic solvents in the elution time T _r in until the elution time T _r in the chromatographic A measured value storage means for storing in association with a specific concentration of the organic solvent,
Change rate storage means for storing a change rate of the elution index T ₀ / T _r with respect to a change in the concentration of the organic solvent in the eluent;
Based on the elution index T ₀ / T _r for the specific concentration stored in the measured value storage means and the change rate of the elution index T ₀ / T _r stored in the change rate storage means, the specific elution index T Concentration calculating means for determining the concentration of the organic solvent in the eluent from which ₀ / T _r0 is obtained; and
Based on the calculation result of the concentration calculation means, the concentration of the organic solvent in the eluent sent to the column so that the elution index T ₀ / T _{r of} the sample becomes the specific elution index T ₀ / T _r0. A control program for a reverse phase liquid chromatograph for causing a computer to function as second concentration control means for outputting a control signal for controlling the control.

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