JP7276809B2 - Preparative Liquid Chromatograph Method, Preparative Liquid Chromatograph Apparatus, and Preparative Liquid Chromatograph Apparatus Control Program - Google Patents

Description

The present invention relates to a preparative liquid chromatograph method, a preparative liquid chromatograph apparatus, and a preparative liquid chromatograph apparatus control program.

A preparative liquid chromatograph device is a device for separating each component in a solution in which multiple components are mixed by the principle of chromatography that utilizes the difference in passage speed of different compounds in the column packing material. is. In recent years, even in such preparative liquid chromatographs, functions have been advanced and automated, and many attempts have been made to achieve efficient preparative separation.

In general, preparative liquid chromatographs are often required to be able to completely separate a plurality of components and to shorten the separation time as much as possible. However, these requirements are inherently contradictory, and an attempt to achieve complete separation tends to lengthen the separation time. Therefore, in order to find separation conditions that satisfy both of these conditions, detailed examination of the separation conditions is required.

In such a preparative liquid chromatograph, the solvent mixing ratio of the influent solvent is often changed over time (hereinafter sometimes referred to as "gradient").
That is, in the initial stage, a solvent composition with high separation ability is used, and over time, the mixture ratio is changed to a solvent composition with a high outflow rate. How to perform such a gradient is a very important factor from the viewpoint of efficient separation in a short time. However, sufficient studies have not been conducted on how to determine such a gradient.

Patent Document 1 discloses a preparative liquid chromatograph using an injector. Furthermore, it describes calculating the polar organic solvent trap volume when using a polar organic solvent and calculating the conditions of chromatography. However, only the correction for the use of a polar organic solvent as the solvent for dissolving the sample has been investigated in such methods, and the general solvent gradient conditions have not been investigated. .

Patent Document 2 discloses a method for determining a gradient curve. However, no consideration is given here to the relationship between the injector and the gradient when the injector is used.

JP 2013-213777 A JP-A-2000-356629

The preparative liquid chromatographic method of the present invention is a chromatographic method that enables efficient separation in a short period of time, and simultaneously achieves the originally difficult objectives of improving the degree of separation and shortening the separation time. is possible.
A preparative liquid chromatograph device and a preparative liquid chromatograph device control program of the present invention are a device and a program for supporting an operator to easily perform the preparative liquid chromatographic method described above. is.

The present invention
A preparative liquid chromatography method using an injector (A) and a separation column (B),
A separation column (B) is connected downstream of the injector (A),
Chromatography is performed while changing the mixing ratio of the solvent,
To change the solvent mixing ratio, the solvent is allowed to flow in at a constant mixing ratio for a certain period of time T from the start of separation, and after a certain period of time T has elapsed, preparative liquid chromatography is performed while changing the solvent mixing ratio,
Let the time t _x for the purification target compound X move from the injector (A) to the separation column (B),
When a constant ratio of the solvent flowing in during a constant time T moves from the injector (A) to the separation column (B) as t _y ,
A preparative liquid chromatography method characterized by setting T so that t _y ≧t _x .

The preparative liquid chromatographic method described above is optimized for T so that the solvent flowing into the injector (A) at time T begins flowing from the injector (A) into the separation column (B) at approximately the same time as _tx . A value may be set.
The preparative liquid chromatography method may set the optimum value of T based on the empty volume of the column according to the size of the column used.

The preparative liquid chromatography method may set T based on the empty volume of the injector according to the size of the injector used.
The preparative liquid chromatography method may include a step of measuring the Rf value of the purification target compound X before preparative liquid chromatography.
The preparative liquid chromatography method has a step of determining T based on the Rf value of the purification target compound X and the time t ₀ ' required for 1 CV (injector volume) of the eluent to flow through the injector. There may be.

The present invention provides a preparative liquid comprising an injector (A), a separation column (B), a computer (C) for controlling the apparatus, and solvent mixture ratio control means (D) controlled by the computer (C). A chromatographic apparatus,
A separation column (B) is connected downstream of the injector (A),
In the change of the mixing ratio by the solvent mixing ratio control means (D), the solvent is allowed to flow in at a constant mixing ratio for a certain time T from the start of separation, and after the lapse of a certain time T, fractionation is performed while changing the solvent mixing ratio. It controls to perform liquid chromatograph for
In the control, the time t _x is taken for the purification target compound X to move from the injector (A) to the separation column (B),
When a constant ratio of the solvent flowing in during a constant time T moves from the injector (A) to the separation column (B) as t _y ,
t _y ≧t _x
It is also a preparative liquid chromatograph characterized in that T is set so as to be

The preparative liquid chromatograph apparatus is configured so that the solvent that has flowed into the injector (A) at time T begins to flow from the injector (A) into the separation column (B) at approximately the same time as tx. A value may be set.
The preparative liquid chromatograph may set the optimum value of T based on the empty volume of the column according to the size of the column used.

The preparative liquid chromatograph may set the optimum value of T based on the empty volume of the injector according to the size of the injector used.
The computer (C) may include input means for inputting the Rf value of the compound X to be purified or the result of thin layer chromatography.
The computer (C) is based on the Rf value input by the Rf value input means or the thin layer chromatography result and the time t ₀ ' required for 1 CV (injector volume) of the eluent to flow through the injector. An inflow time calculation means for calculating the time _tx for the target compound X to move from the injector (A) to the separation column (B) may be provided.

The present invention provides a preparative liquid comprising an injector (A), a separation column (B), a computer (C) for controlling the apparatus, and solvent mixture ratio control means (D) controlled by the computer (C). A program for controlling a chromatographic apparatus,
A separation column (B) is connected downstream of the injector (A),
In the change of the mixing ratio by the solvent mixing ratio control means (D), the solvent is allowed to flow in at a constant mixing ratio for a certain time T from the start of separation, and after the lapse of a certain time T, fractionation is performed while changing the solvent mixing ratio. It controls to perform liquid chromatograph for
In this control, the time t _x for the purification target compound X to move from the injector (A) to the separation column (B)
When a constant ratio of the solvent flowing in during a constant time T moves from the injector (A) to the separation column (B) as t _y ,
It is also a preparative liquid chromatograph control program characterized by setting T so that t _y ≧t _x and performing control.

The program for controlling the preparative liquid chromatograph device is designed so that the solvent that has flowed into the injector (A) at time T will start flowing from the injector (A) into the separation column (B) at approximately the same time as _tx . The optimum value of T may be set to .
The preparative liquid chromatograph control program may set the optimum value of T based on the empty volume of the column according to the size of the column used.

The preparative liquid chromatograph apparatus control program may set T based on the empty volume of the injector according to the size of the injector to be used.
The preparative liquid chromatograph apparatus control program may have means for inputting the Rf value of the purification target compound X before preparative liquid chromatograph is performed.
The program for controlling the preparative liquid chromatograph device determines T based on the Rf value of the compound X to be purified and the time t ₀ ' required for 1 CV (injector volume) of the eluent to flow through the injector. It may have

The preparative liquid chromatography method of the present invention enables an operator to perform efficient separation in a short period of time. Furthermore, the preparative liquid chromatograph device and the preparative liquid chromatograph device control program of the present invention can assist the operator in performing efficient separation.

1 is a schematic diagram showing an example of the configuration of a preparative chromatograph apparatus of the present invention; FIG. It is a schematic diagram which shows an example of the gradient method of this invention. It is a schematic diagram for demonstrating the method to measure Rf by thin layer chromatography. 1 is a diagram showing the results of chromatography in Example 1. FIG. 4 is a diagram showing the results of chromatograph of Comparative Example 1. FIG. FIG. 4 is a diagram showing the chromatographic results of Example 2. FIG. FIG. 10 is a diagram showing the chromatographic results of Comparative Example 2;

The present invention is characterized in that, in a preparative liquid chromatographic method using an injector (A) and a separation column (B), a gradient is performed according to a method specifically described in detail below. have. In other words, the present invention was completed by the present inventor's study and finding that separation can be performed with high accuracy when specific conditions are set for changes in the solvent mixture ratio.

More specifically, the solvent mixing ratio is changed so as to satisfy the following conditions.
(1) A solvent having a constant mixing ratio is allowed to flow in for a certain period of time T from the start of separation, and preparative liquid chromatography is performed while changing the solvent mixing ratio after the lapse of the certain period of time T.
(2) Time t _x for the purification target compound X to move from the injector (A) to the separation column (B), and a certain ratio of the solvent flowing in during a certain time T from the injector (A) to the separation column (B). When the moving time is t _y ,
T is set so that t _y ≧t _x .

In other words, at the same time as or later than when the purification target compound X moves from the injector (A) to the separation column (B), the solvent flowing into the injector at a certain time T moves from the injector (A) to the separation column ( B) is carried out by changing the solvent mixture ratio.

Usually, when performing a gradient in preparative liquid chromatography, a solvent with relatively low polarity and high resolution is first used, and this is mixed with a solvent with high polarity and low resolution. It is. First, a solvent with high separation performance is used to separate the target compound to some extent, and when the separation is completed to some extent, a solvent with high separation performance is used to shorten the time until it flows out.

When performing such separation, it is necessary to perform sufficient separation at the initial stage. For this purpose, the present invention was completed by discovering that it is necessary to perform a gradient under the condition (2) described above.

At t _x after a certain period of time has passed since the start of introduction of the eluent, the purification target compound X that the operator wishes to obtain with high accuracy and the solvent at the time of starting the gradient are almost simultaneously released from the injector (A) into the separation column ( More preferably, the gradient conditions are set to flow into B). In this way, if they are substantially the same, it is preferable in that the separation can be completed in the shortest time while ensuring good separation accuracy.

Here, "substantially the same" means that there may be a difference of 10% before and after the time _tx . That is, it is not always necessary to make them exactly the same, and some deviation is allowed.

In the chromatographic method of the present invention, an injector (A) and a separation column (B) are used.
In the present invention, the injector (A) has a cylinder filled with a filler and uses a dry filler having adsorption capacity as the filler. In chromatography, a sample to be separated is injected into such an injector (A), and the injector (A) is installed in the device, whereby the sample can be introduced into the channel.

The dry filler is filled in the cylinder in a dry state without containing a solvent, and the sample solution is introduced in the dry state. By introducing the sample liquid in a dry state and then introducing the solvent, the polar organic solvent is efficiently adsorbed to the filler in the injector cylinder.

In the present invention, a separation column (B) is used. As such a separation column (B), a general column manufactured and sold for a general preparative liquid chromatograph can be used. The separation column (B) also has a cylinder filled with a packing material, and is filled with a dry packing material having an adsorptive capacity.

A schematic diagram showing an example of an apparatus used in the preparative liquid chromatography method of the present invention is shown in FIG. In addition, the preparative liquid chromatography method of the present invention is not limited to the method using the apparatus shown in _FIG . It doesn't matter if it's something.

In FIG. 1 the injector (A) is indicated as 12 and the separation column (B) is indicated as 1 .
The figure shows two kinds of organic solvents 3A and 3B. The introduction of these different organic solvents is controlled by the pump 13, and is introduced into the channel as a mixed solvent having a predetermined mixing ratio in accordance with instructions from the computer 5 as required.
In FIG. 1, a flow path is formed from injector 12→separation column 1→sample acquisition means 2, and the inflow solvent flows along this flow path.

A sample to be separated is a mixed solution containing multiple components, and such a sample is introduced into injector (A) 13 . In this case, it is preferable that the injector (A) has a cartridge form, and after the sample is introduced into the cylinder, it is set in the channel, and then the solvent starts to flow.

The eluate eluted from the separation column 1 is sequentially obtained by the sample obtaining means 2 . That is, it is acquired in the test tube for each outflow time.

As described above, in the present invention, preparative liquid chromatography is performed while performing a solvent gradient. A typical gradient pattern is shown in FIG. In such a gradient pattern, the solvent is allowed to flow in without changing the solvent mixture ratio for a certain period of time (between 0 and T), and then the solvent mixture ratio is changed at a constant rate.

From this T, the solvent is introduced by the pump 13 while sequentially changing the mixing ratio of each solvent. Then, the mixed solvent that has flowed in from the pump at time T moves from the injector (A) to the separation column (B) after a certain period of time has elapsed.

On the other hand, the sample introduced into the injector moves in the injector while interacting with the adsorbent filler. Therefore, it moves through the injector at a speed slower than that of the solvent.
Thus, at some point the later introduced solvent will catch up with the earlier introduced sample. That is, when the gradient is performed in the pattern shown in FIG. 2, the mixed solvent introduced at time T will catch up with the purification target compound X at some time. Controlling this catch-up point is important in the present invention.

In the present invention, the purification and separation target compound X is a target compound that the operator wishes to obtain as a highly pure compound.
In general chemical experiments in laboratories, the desired compound is clearly present, chemical reactions are carried out to obtain the desired compound, and then purification is carried out in most cases.
Therefore, in the preparative liquid chromatographic method of the present invention, it is not always necessary to perform precise separation for all components. will be achieved. A compound that the operator wishes to obtain with high accuracy is defined as a purification target compound X, and the time required for this compound to move from the injector (A) to the separation column (B) is defined as _tx .

Furthermore, in the present invention, the time for the solvent introduced into the injector at time T described above to move from the injector (A) to the separation column (B) is defined as t _y . Since the solvent does not interact with the stationary phase in the injector, _ty is determined by the relationship between the flow rate of the liquid and the empty volume of the injector. The present invention is characterized in that there is a relationship of t _y ≧t _x between them.

In order to perform the preparative liquid chromatograph of the present invention as described above, it is preferable to create an appropriate gradient pattern corresponding to the sample to be separated.
More specifically, how to set the time T to start changing the solvent mixture ratio from a constant mixture ratio is important.

Such setting of T is preferably performed according to the column size and the injector to be used. That is, since the empty volume of the column and the empty volume of the injector are values closely related to t _y and t _x , it is preferable to set T so as to achieve predetermined t _y and t _x . .

In the present invention, a gradient pattern is created according to the sample to be separated so as to achieve the above-mentioned purpose, and the solvent mixing ratio is controlled based on this.

How to determine T shown in FIG. 2 is important in the creation of the gradient pattern in the present invention. In carrying out the present invention described above, it is preferable to calculate T such that t _x =t _y (hereinafter referred to as Ts) and set T in the gradient pattern based on this. That is, if T _s is known, it is easy to choose T such that t _x t _y ≧t _x .

Although the method for determining such _Ts is not particularly limited in the present invention, as a specific method, for example, the Rf value is obtained by performing thin layer chromatography or column chromatography on the sample to be measured in advance. On the other hand, the time t ₀ ′ required for 1 CV (injector empty volume) of the eluent to flow through injectors of various sizes can be measured, and T _s can be calculated based on these.

An example of such a calculation method is shown below.
The method of thin-layer chromatography performed in a preferred embodiment of the present invention is not particularly limited, and can be performed by a generally known method. As a developing solvent for thin-layer chromatography, it is most preferable to use a mixed solvent having a constant solvent mixture ratio B1 used from time=0 to T _s in the gradient of FIG.

Also, the Rf value in B1 may be calculated by performing thin-layer chromatography at other mixing ratios and using a known formula or the like based on the results.

FIG. 3 shows a schematic diagram of thin-layer chromatography. In FIG. 3, the Rf of the purification target compound X is
Rf = Ls/Lo
Calculated by Confirmation of which spot is the compound X to be purified is not particularly limited, and can be performed by any known method.

First, in chromatography, an eluent is allowed to flow at a flow rate v (volume/time). Let 1 t ₀ ′ be the time required for 1 CV (the empty volume of the injector) of the eluent to flow through the injector.
From the definition above,
1CV= _vt0 '
is.

For R _f measured by the method described above, if the moving speed of the sample itself is represented by volume and v' (volume / time),
v′=Rfv
is.
Also, a volume of CV/ _Rf of eluent must flow for the sample to pass through the injector.

Now, when the liquid is sent for the time T _s , the movement position volume V of the sample is
V=v'Ts
=vTsRf
is.

If the gradient is started after pumping for time T _s , the sample receives a further volume V of solution (ie, the volume of eluent remaining above the sample in the injector).
If the time T' is when the gradient reaches the sample, then the eluent solution W received by the sample at T' is
W= _vTs +V
=vt+RfvT _s
=(1+Rf)vT _s
becomes.
On the other hand, the sample when W=CV/Rf passes through the injector and onto the separation column. Therefore,
W=(1+Rf)vTs=CV/Rf= _vt0 '/Rf
The relational expression of is established.
Therefore,
Ts= _t0 '/(Rf(1+Rf))
becomes a relationship.

Based on the above relational expression, Ts can be calculated from Rf.
For example, if Rf = 0.2, from the above formula,
Ts= _4.2t0 '
can be calculated, and once t ₀ ′ is known, the above equation allows the implementation of the present invention.

In addition, since t ₀ ′ is a value closely related to the empty volume of the injector, it is possible to calculate Ts as described above according to the empty volume of the injector to be used and find the optimum gradient condition. can.

The preparative liquid chromatograph apparatus of the present invention has functions necessary to support execution of the above-described chromatograph. That is, it comprises an injector (A), a separation column (B), a computer (C) for controlling the apparatus, and solvent mixing ratio control means (D) controlled by the computer (C). In (C), the function of controlling the solvent mixture ratio as described above is provided.

By using such an apparatus, the preparative liquid chromatographic method described above can be easily performed with less labor by performing automatic calculation and management by a computer. Such a preparative liquid chromatograph is also one aspect of the present invention.

Such preparative liquid chromatograph apparatus is controlled by computer 5 . That is, the computer controls the solvent mixing ratio (that is, the mixing ratio of the two solvents 3A and 3B in FIG. 1), which is a feature of the present invention.

Control of the preparative liquid chromatograph apparatus as described above by computer can be performed by general programming. Such a program may be stored in a personal computer or the like, or may be stored in a server and controlled via the Internet.

Furthermore, the preparative liquid chromatograph apparatus of the present invention preferably comprises input means for inputting the Rf value or thin layer chromatographic result of the sample to be separated. As described above, in the present invention, when determining T, a calculation method using the Rf value is preferred. Therefore, in order to calculate the T value according to such a method, it is preferable to input the Rf value or the thin layer chromatographic result. By inputting the thin layer chromatographic result, the Rf value can be easily calculated therefrom. For this reason, in the case of having means for inputting thin layer chromatographic results, it is preferable to also have Rf value calculating means for calculating the Rf value from the results.

A method of inputting such an Rf value is not particularly limited, and any known method can be used. For example, a method of inputting the Rf value after calculating the Rf value, a method of acquiring an image by a method such as photography or scanning of a thin layer chromatographic plate and calculating the Rf value by image analysis, a method of manually inputting the thin layer chromatography A method of inputting an image of a graph board and calculating the Rf value based on the result can be used.

Furthermore, the control of the solvent mixing ratio control means (D) in the preparative liquid chromatograph apparatus determines T by the method described above, controls the operation of the pump based on the determined T, and It is preferable to control the mixing ratio of the solvent.

A control program for a preparative liquid chromatograph apparatus of the present invention is a program for performing control in a preparative liquid chromatograph apparatus.
That is, in carrying out the preparative liquid chromatograph of the present invention as described above, it is preferable to use a computer to perform necessary calculations and device control. A program having the necessary requirements for performing such control is also one aspect of the present invention.

EXAMPLES The present invention will be specifically described below based on examples.
A preparative liquid chromatograph was performed using the apparatus shown in FIG. As a sample to be separated, a dichloromethane solution of a mixture of butyl p-hydroxybenzoate and methyl p-hydroxybenzoate was used.
Further, the sample after passing through the separation column (B) was detected by an ultraviolet absorption detector.

For separation, a mixed solvent of hexane/ethyl acetate was used as an eluent. At the initial stage of starting separation, an eluent with a mixture ratio of hexane/ethyl acetate=83/17 was used, and the ratio of ethyl acetate was gradually increased after a certain period of time. The change pattern of this ratio was the same in all Examples and Comparative Examples, and the solvent mixture ratio was changed at a constant rate so as to finally achieve hexane/ethyl acetate=62:28.

Before performing the above preparative liquid chromatography, thin-layer chromatography with silica was performed using a mixed solvent of hexane/ethyl acetate=83/17. As a result, butyl p-hydroxybenzoate had an Rf of 0.22 and methyl p-hydroxybenzoate had an Rf of 0.20. T was calculated based on this Rf value and t ₀ ′ of the injector used. Methyl p-hydroxybenzoate was used as compound X in the calculation of T.

A small amount of toluene was added to the solvent in the preparative liquid chromatography. Toluene is a component that is not adsorbed by the filler, and was added as an indicator of the position of the solvent at the start of the inflow.

(Example 1)
A sample was injected into the injector, and preparative liquid chromatography was performed using the apparatus shown in FIG.
Then, based on the Rf value of the compound X described above and the passage time of the solvent through the injector, a gradient is performed based on T (4.16 minutes) such that ty = tx, which is calculated by the method described above. Liquid chromatography was performed.
FIG. 4 shows the result of confirming the separation state by the gradient state and the detector. It is clear that good separation can be achieved by this, and the separation is completed in a short time.

(Comparative example 1)
Preparative liquid chromatography was performed in the same manner as in Example 1 except that T=0. FIG. 5 shows the result of confirming the separation state by the gradient state and the detector. Comparing this result with Example 1, it is clear that the two peaks overlap and the separation is not complete.

(Example 2)
Preparative liquid chromatography was performed in the same manner as in Example 1, except that T was set to 11 minutes. FIG. 6 shows the result of confirming the separation state by the gradient state and the detector. Good separation could be achieved also from this result. However, in comparison with Example 1, although the two peaks are separated, the peaks are broadened, and the eluent in which the sample is dissolved continues to flow out over a long period of time.
As a result, the chromatographic time is lengthened, and the time required for concentrating the effluent is also lengthened.

(Comparative example 2)
A preparative liquid chromatograph was performed in exactly the same manner as in Example 1, except that the injector was not used. FIG. 7 shows the result of confirming the separation state by the gradient state and the detector. Comparing this result with Example 1, it is clear that the two peaks overlap and the separation is not complete.

From the results of the above examples and comparative examples, it has become clear that separation can be carried out under optimum separation conditions by performing chromatography according to the method of the present invention.

The preparative liquid chromatograph method, preparative liquid chromatograph apparatus, and preparative liquid chromatograph apparatus control program of the present invention can be suitably used in the separation of mixtures in laboratories in the field of chemistry.

1 column 2 fraction collector 5 controller 11 eluent 12 injector 13 pump 21 compound X spot

Claims (15)

A preparative liquid chromatography method using an injector (A) and a separation column (B),
A separation column (B) is connected downstream of the injector (A),
Chromatography is performed while changing the mixing ratio of the solvent,
To change the solvent mixing ratio, the solvent is allowed to flow in at a constant mixing ratio for a certain period of time T from the start of separation, and after a certain period of time T has elapsed, preparative liquid chromatography is performed while changing the solvent mixing ratio,
The time tx for the purification target compound X to move from the injector (A) to the separation column (B),
When a constant ratio of the solvent flowing in during a constant time T is moved from the injector (A) to the separation column (B) as ty,
Set T so that ty≧tx ,
The optimum value of T is set so that the solvent that has flowed into the injector (A) at time T begins to flow from the injector (A) into the separation column (B) at approximately the same time as tx.
A preparative liquid chromatography method characterized by: 2. The preparative liquid chromatography method according to claim 1 , wherein the optimum value of T is set based on the empty volume of the column according to the size of the column used. 3. The preparative liquid chromatography method according to claim 1 , wherein T is set based on the empty volume of the injector according to the size of the injector used. 4. The preparative liquid chromatography method according to claim 1, 2 or 3, further comprising a step of measuring the Rf value of said compound X to be purified before preparative liquid chromatography. 5. The component according to claim 1, 2, 3 or 4, further comprising the step of determining T based on the Rf value of the compound to be purified X and the time t0' required for 1 CV (injector volume) of the eluent to flow through the injector. Preparative liquid chromatographic method. A preparative liquid chromatograph device comprising an injector (A), a separation column (B), a computer (C) for controlling the device, and solvent mixing ratio control means (D) controlled by the computer (C) There is
A separation column (B) is connected downstream of the injector (A),
In the change of the mixing ratio by the solvent mixing ratio control means (D), the solvent is allowed to flow in at a constant mixing ratio for a certain time T from the start of separation, and after the lapse of a certain time T, fractionation is performed while changing the solvent mixing ratio. It controls to perform liquid chromatograph for
In the control, the time tx for the purification target compound X to move from the injector (A) to the separation column (B),
When a constant ratio of the solvent flowing in during a constant time T is moved from the injector (A) to the separation column (B) as ty,
T is set so that ty≧tx,
The optimum value of T is set so that the solvent that has flowed into the injector (A) at time T begins to flow from the injector (A) into the separation column (B) at approximately the same time as tx.
A preparative liquid chromatograph, characterized by: 7. The preparative liquid chromatograph apparatus according to claim 6 , wherein the optimum value of T is set based on the empty volume of the column according to the size of the column used. 8. The preparative liquid chromatograph apparatus according to claim 6 or 7, wherein the optimum value of T is set based on the empty volume of the injector according to the size of the injector to be used. 9. The preparative liquid chromatograph apparatus according to claim 6, 7 or 8, wherein the computer (C) comprises input means for inputting the Rf value of the compound X to be purified or the thin layer chromatographic result. The computer (C), based on the Rf value input by the Rf value input means or the thin layer chromatographic result and the time t0' required for 1 CV (injector volume) of the eluent to flow through the injector, the compound to be purified 10. A preparative liquid chromatograph according to claim 6, 7, 8 or 9 , further comprising inflow time calculating means for calculating a time tx for X to move from the injector (A) to the separation column (B). A preparative liquid chromatograph device comprising an injector (A), a separation column (B), a computer (C) for controlling the device, and solvent mixing ratio control means (D) controlled by the computer (C) A program for controlling
A separation column (B) is connected downstream of the injector (A),
In the change of the mixing ratio by the solvent mixing ratio control means (D), the solvent is allowed to flow in at a constant mixing ratio for a certain time T from the start of separation, and after the lapse of a certain time T, fractionation is performed while changing the solvent mixing ratio. It controls to perform liquid chromatograph for
In this control, the time tx for the purification target compound X to move from the injector (A) to the separation column (B)
When a constant ratio of the solvent flowing in during a constant time T is moved from the injector (A) to the separation column (B) as ty,
T is set so that ty≧tx, and control is performed ,
The optimum value of T is set so that the solvent that has flowed into the injector (A) at time T starts flowing from the injector (A) into the separation column (B) at approximately the same time as tx. A program for controlling the liquid chromatograph system. 12. The program for controlling a preparative liquid chromatograph device according to claim 11 , wherein the optimum value of T is set based on the empty volume of the column according to the size of the column used. 13. The preparative liquid chromatograph apparatus control program according to claim 11 or 12 , wherein T is set based on the empty volume of the injector according to the size of the injector to be used. 14. The program for controlling a preparative liquid chromatograph device according to claim 11, 12 or 13, further comprising means for inputting the Rf value of said compound X to be purified before conducting preparative liquid chromatograph. 15. The separation according to claim 11, 12, 13 or 14 , further comprising the step of determining T based on the Rf value of the compound to be purified X and the time t0' required for 1 CV (injector volume) of the eluent to flow through the injector. A program for controlling the liquid chromatograph system.

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