JPH08136522A - Packing method of column for liquid chromatography - Google Patents

Abstract

PURPOSE: To produce a high performance column by filling a column for liquid chromatography uniformly with a packing agent. CONSTITUTION: A fluid is fed through a movable plug 1 into a column 2 thus stirring a packing agent slurry 3 in the column 2. The packing agent slurry 3 in the column 2 is stirred sufficiently by the fluid fed through the movable plug 1 thus preventing the packing agent from settling. Consequently, the packing agent can be laminated continuously from the side opposite to the movable plug 1 to form a uniform packing agent layer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for packing a packing material into a column for liquid chromatography used for the purification of high-purity chemicals such as pharmaceuticals and fine chemicals.
In particular, it relates to a method for uniformly filling a packing material in the column to prepare a high performance column.

[0002]

2. Description of the Related Art Recently, liquid chromatography has been used for natural products, fermentation products, culture products by gene recombination, etc.
As a means for separating and purifying a target substance in a synthetic reaction product at a target purity and a purification rate, it has come to be widely used for industrial purposes by pharmaceutical companies, chemical companies and the like.

Conventionally, packing of a packing material into a liquid chromatography column has been carried out by the following method.

That is, first, as shown in FIG. 2A, a column 2 provided with a movable stopper 1 is used, and as shown in FIG.
A slurry 3 prepared by mixing a filler with a solvent is charged into the column 2. Then, as shown in FIG. 2C, the upper lid 4 having a perforated plate and provided with a solvent outlet is attached to the upper part of the column, and the movable stopper 1 is pushed up to push out the solvent from the solvent outlet of the upper lid 4. As shown in (d), the filling layer 5 of the filler is formed under pressure. That is, by the movement of the movable stopper 1, a uniform filling layer is laminated from the side opposite to the movable stopper, and after the filling is completed, the filling layer 5 by the movable stopper 1 is stacked.
Maintain the pressurization of. This filling method is described in JP-B-58-202.
No. 84, and this packing device is widely known as an axial compression type column.

On the other hand, a method of using a slurry in which the filler hardly sediments in order to prevent the filler from settling during the filling operation and form a uniform packed layer is known as an equilibrium slurry filling method. This method includes a method using a solvent having a high viscosity and a method using a solvent having a specific gravity close to that of the filler. Among these, as an example of using a solvent having high viscosity, a method using a methanol / ethylene glycol-based solvent (J. Li.
q. Chromatogr. 2 , 129, 1979) and the like. As an example of using a solvent having a specific gravity close to that of the packing material, there is a method using a tetrabromethane / carbon tetrachloride system (“Selection and usage of column packing material” Kodansha Scientific p26 1983). These methods have been introduced as slurries for making small packed columns such as analytical columns.

Incidentally, for packing a packing material having a large settling speed and a large particle size, this is conventionally used as a slurry, and there is no movable stopper freely movable in the column while stirring the slurry with a stirrer. A method of filling a column with a slurry pump is generally used.

[0007]

In the conventional packing method shown in FIGS. 2 (a) to 2 (d), the packing material having a large particle size has a high sedimentation speed in the slurry, and the packing is performed after the slurry is put into the column. By the end, the filler settles on the movable stopper. Therefore, in the formed packed layer, there are two packed layers, that is, the packed layer laminated from the side opposite to the movable plug and the packed layer that naturally precipitates on the movable plug. Further, since the packed bed settling on the movable plug pushes it up by the movable plug, distortion may occur in the settled packed bed. For this reason, the packed bed produced by this method becomes a non-uniform packed bed, and the column performance (theoretical plate number, peak shape) is poor.

Further, as an equilibrium slurry filling method for preventing the settling of the filler in the slurry, a method of increasing the viscosity of the solvent with liquid paraffin, ethylene glycol or the like, or using an organic halogen type solvent having a specific gravity close to that of the filler is used. After filling, it is necessary to completely replace the solvent with the solvent used for purification by liquid chromatography. In this case, there is a problem that it takes time and labor for the replacement, and further, it takes a lot of time and labor to confirm that the replacement is complete. Moreover, since a solvent for filling and a solvent for purification are required, the solvent cost is high and it is economically disadvantageous, and in particular, in recent years, organic halogen-based solvents tend to be refrained from the viewpoint of environmental problems. is there. From such a thing,
It is desirable to use a solvent having the same or similar properties as the refining solvent for the filling slurry, and the equilibrium slurry filling method using a dispersion medium different from the refining solvent cannot be said to be an industrially advantageous method.

By the way, a method of filling a column having no movable stopper with a slurry pump while agitating a slurry of a filler having a large particle size is a method capable of producing a column having relatively good column performance.

However, in a liquid chromatography column, not only the packing workability of the packing material but also the packing workability is an important requirement. That is, for example, a silica gel coarse particle size filler is a filler having a high sedimentation speed in a slurry, but is often used as a pretreatment or a disposable filler because it is relatively inexpensive, and therefore, it can be filled and extracted. The frequency of operations is very high.

In the case of a column without a movable stopper, when removing the packing material, the lid at the bottom of the column is usually opened to extract the packing material. However, when the column becomes large, the weight of the lid becomes heavy and the operation of extracting the packing material becomes difficult. It is regarded as a problem that workability becomes extremely poor.

On the other hand, in the case of a column having a movable stopper, the movable stopper can be raised and the packing material can be extracted from the upper part of the column. Even if the column becomes large in size, this operation
The upper lid may be removed with a chain block or the like, and the filler can be gradually pushed up by controlling the rising speed of the movable plug, so that the withdrawal work can be performed very easily and safely.

Therefore, in consideration of not only the filling operation of the packing material but also the extraction operation, it is preferable to use a column having a movable stopper.

The present invention has been made in view of the above-mentioned conventional circumstances. A column having a movable stopper is used, and a packed slurry of a solvent having the same or similar property as that of the purifying solvent is charged to obtain a uniform mixture. An object of the present invention is to provide a packing method for a column for liquid chromatography which forms a packed bed.

[0015]

According to a first aspect of the present invention, there is provided a liquid chromatography column packing method, wherein a slurry prepared by mixing a packing material with a solvent is movable in the column from one end side to the other end side. After inserting the movable stopper into a column equipped with a stopper in a state where the movable stopper is retracted to one end side of the column, a lid having a solvent outlet and a perforated plate is attached to the other end side of the column, and then the movable stopper is attached. In the method of packing a column for liquid chromatography, wherein a packing layer of a packing material is formed in the column by advancing the plug to the other end side, the movable plug is made porous, and further the movable plug is placed inside the column. It is characterized in that a fluid is supplied to stir the slurry in the column.

A method for packing a column for liquid chromatography according to a second aspect is the method according to the first aspect, wherein the gas supply is started immediately before or after the slurry is charged into the column, and then the advance of the movable stopper is started. It is characterized in that the supply of the gas is stopped immediately before, the supply of the liquid is started, and the supply of the liquid is continued until the filling is completed.

The present invention will be described in detail below with reference to the drawings. 1 (a) to 1 (e) are cross-sectional views showing a method of packing a column for liquid chromatography according to an embodiment of the present invention. Members having the same functions as those shown in FIG. I am doing it.

According to the method of the present invention, during the filling operation, gas or liquid is supplied to the packing slurry in the column through the movable stopper to stir the slurry, so that the packing material in the slurry is allowed to settle. To prevent.

In the practice of the present invention, when gas is supplied from the movable plug side, if the gas is ventilated after the packed layers have started to be stacked, the gas may disturb the stacked packed layers. Also,
If the liquid is passed from the movable stopper side before the filler slurry is put into the column and before the upper lid is attached, the slurry may overflow from the upper part of the column.

In order to solve such a problem, in carrying out the present invention, as shown in FIGS. 1 (a) to 1 (e),
It is preferable to switch between ventilation and liquid passage according to the following procedure.

First, immediately before (or immediately after) charging the filler slurry 3 into the column 2, aeration of gas is started via the movable plug 1 (FIG. 1 (a)), and the slurry 3 is placed in the column 2 in this state. throw into. The slurry charged into the column is bubbled by the bubbles 6 due to aeration, the packing material is in a good floating state, and sedimentation is prevented (FIG. 1 (b)).

After the slurry 3 has been charged, the slit upper lid 4 is attached in a ventilated state (see FIG. 1).
(C)).

After that, the ventilation from the movable stopper 1 is stopped to switch to the liquid passage, and the movable stopper 1 is started to rise (FIG. 1 (d)). The temperature is raised to complete the filling (FIG. 1 (e)). After the filling is completed, the liquid flow is stopped.

In the present invention, any gas may be used as long as it does not affect the filler or solvent, and in general, nitrogen, air or the like is used. The gas aeration rate is not particularly limited as long as the slurry in the column is in a sufficiently agitated state, but is usually 0.02 to 0.2 Nl / column unit cross-sectional area.
It is set to about min / cm ² .

As the liquid, the same solvent as the filler slurry is usually used. The flow rate of this solvent is also not particularly limited as long as the slurry in the column is in a sufficiently stirred state, but in general, the linear velocity is about 2 to 10 m / hr. It should be noted that this liquid flow rate is required to be smaller than the solvent outflow rate from the upper lid of the column.

In the present invention, the solvent used for the filler slurry and the concentration of the filler slurry to be filled may be the same as those in the conventional method.

The method of the present invention as described above is extremely effective especially for filling coarse particle size fillers of silica gel which are widely used in industry.

[0028]

The fluid supplied from the movable stopper can sufficiently stir the filler slurry in the column to prevent the packing from settling. Therefore, the filler can be filled as a continuous and uniform filling layer laminated from the side opposite to the movable stopper.

According to the method of claim 2, a better filling operation can be performed.

[0030]

EXAMPLES The present invention will be described more specifically with reference to Examples and Comparative Examples below.

Example 1 Filling was performed according to the method shown in FIGS. 1 (a) to 1 (e). The column size, packing material, solvent and slurry concentration used are as follows.

Column size: inner diameter 300 mm, length 1000 mm Filler: silica gel 230/400 mesh (Merc
k company) Solvent: n-hexane / ethyl acetate mixed solution Slurry concentration: 300 g-silica gel / l-solvent In filling, first, supply of nitrogen is started via a movable stopper immediately before the slurry is put into a column. (Supply speed 100
Nl / min), and then 130 liters of the slurry was added thereto, and the upper lid was attached. Immediately before raising the movable stopper, the supply of nitrogen is stopped and the supply of the same solvent as the solvent of the slurry is started (supply amount 5 liters / mi).
n), and then the movable stopper was raised to perform filling (solvent outflow rate was 10 l / min). After completion of the filling, the supply of the solvent from the movable stopper was stopped.

The standard performance of the packed column thus obtained was measured under the following measurement conditions. The results are shown in Fig. 3 (a) and Table 1.

Mobile phase: n-hexane / ethyl acetate mixed solvent Flow rate: 3.6 m / h (flow rate 4.2 liters / min) Detection: UV 254 nm Temperature: 25 ° C. Sample: Toluene Comparative Example 1 Nitrogen from movable stopper and 2 (a) without supplying solvent.
Filling of the filler was performed according to the conventional method shown in (d).
The column, packing material, solvent and slurry concentration used were the same as in Example 1.

The standard performance of the packed column thus obtained was measured in the same manner as in Example 1, and the results are shown in FIG. 3 (b) and Table 1.

[0036]

[Table 1]

From Table 1 and FIG. 3, in Example 1, Comparative Example 1
As compared to the method, a uniform packed bed can be formed, so column performance is good and both theoretical plate number and peak shape are
It is clear that very good results are obtained.

[0038]

As described above in detail, according to the method for packing a column for liquid chromatography of the present invention, a column having a movable stopper is used to prevent the packing material from settling in the slurry during packing and to make it uniform. It is possible to easily and efficiently form a different packed bed to form a column having excellent column performance. Therefore, according to the present invention, it is not necessary to use a special solvent as the filling solvent,
Since a solvent having the same or similar properties as the solvent for purification of liquid chromatography can be used, the replacement of the solvent after filling is unnecessary or small. Therefore, the time and labor for the replacement can be eliminated, and the solvent cost can be reduced. Even with a large column or a packing material having a high sedimentation speed in the slurry, a high-performance column can be prepared by forming a good packed bed with a column having a movable stopper. Since the column having the movable stopper is used, the work of extracting the packing material is facilitated and the safety is improved. Such an effect is produced.

According to the method of claim 2, a better filling operation can be performed.

[Brief description of drawings]

FIG. 1 is a cross-sectional view showing an example of a method for packing a liquid chromatography column according to the present invention.

FIG. 2 is a cross-sectional view showing a conventional filling method.

FIG. 3 is a graph showing analysis results in Example 1 and Comparative Example 1.

[Explanation of symbols]

 1 Movable stopper having a perforated plate 2 Column 3 Slurry 4 Perforated plate and upper lid with solvent outlet 5 Packed layer

Claims (2)

[Claims] 1. A column provided with a movable stopper capable of moving a filler mixed with a solvent into a column from one end side to the other end side, and retracting the movable stopper to the one end side of the column. After loading the column, a lid having a solvent outlet is attached to the other end side of the column, and then the movable stopper is advanced to the other end side to form a packed bed of the packing material in the column. In a method for packing a column for liquid chromatography to be formed, the movable stopper is made porous, and a fluid is supplied from the movable stopper into the column to stir the slurry in the column. How to fill the column. 2. The method according to claim 1, wherein the gas supply is started immediately before or after the slurry is charged into the column, and thereafter, the gas supply is stopped immediately before the advance of the movable stopper is started, and the liquid is supplied. The method for packing a column for liquid chromatography, comprising: starting the supply of the liquid and continuing the supply of the liquid until the packing is completed.