JPS6214059A - Separation refining system appatatus for substance by liquid chromatography - Google Patents

Abstract

PURPOSE:To obtain a industrially practicable material parceling apparatus with a high reliability, by automatically correcting deviation generated in the set condition caused in the ongoing chromatographic process. CONSTITUTION:To introduce a solvent C into a column 1 of a chromatographic separation column system 10 from a solvent feeder 16. A liquid stock feeder 11 is controlled with a controller 35 to bring the liquid stock (A) into a column 1, where chromatographic separation and development of component substances are done in the liquid stock (A). The substances of an agent packed in the column 1 obtained by the chromatographic separation and development are isolated sequentially and flows out to a piping 28 together with the solvent C, sampled discriminatively with a fraction collator 29 and introduced into a concentrator 30 to be extracted as concentrate. On the other hand, the flow rate of the solvent flowing out to the piping 28 is inputted into the unit 35 with a flow rate measuring device 27 to automatically adjust the unit 16 so that the flow rate of the solvent to be introduced into the column 1 can be always kept at a reference value. The solvent to be shunted from a branch tube 31 enters a component substance parceling state measuring monitor 33 and a salt concentration measuring device 34, the measured value is inputted into the unit 35 and thus, the unit 16 is adjusted automatically.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Object of the Invention [Field of Industrial Application] The present invention relates to a system for separating and purifying substances by liquid chromatography.

[Conventional technology]

Liquid chromatography uses multiple types of substances (
To separate (separate and purify) one, several, or all of the target component substances from a crude raw material liquid (original sample) that contains a mixture of inorganic substances, organic substances, biochemical substances, etc. This is one of the effective means.

The basic operation will be briefly explained using the fourth drawing.

(a) Chromatographic separation and development operation of each component substance A solid packing material 2 is packed inside as a stationary phase, and an inlet 3 is placed in a column 1 having a fluid inlet 3 at one end and an outlet 4 at the other end.
Then, a predetermined amount of raw material solution A whose concentration is appropriately adjusted as necessary is introduced, and a developing solvent B (phosphate buffer with a predetermined salt concentration, KCI solution, φNaCl solution, etc.) is introduced.

Reference numerals 5 and 6 indicate filter members disposed at the upper and lower ends of the column.

Then, each component substance contained in the raw material liquid A flowing through the solid packing material 2 in the column in the four directions of the outlet port will change due to differences in adsorption properties and distribution coefficients to the solid packing material 2. A difference in movement speed occurs between the amounts, and as a result, an adsorption zone of each component substance is formed on the column packing material 2 along the flow direction of the raw material liquid, where each component substance is separated from each other.

That is, the component substances are chromatographically separated from each other.

Even after introducing a predetermined amount of raw material liquid A, the introduction of developing solvent B is continued for an appropriate period of time, so that the separation and adsorption zone of each component substance relative to filler 2 moves in the flow direction of solvent B as a whole. The distance between each adsorption zone opens (expands),
Separate better.

(b) Preparative operation of each chromatographically separated substance Once the separation and adsorption zone development of each component substance on the column packing material 2 has progressed appropriately, mutual separation adsorption is performed from the inlet 3 of the column 1 to the column packing material 2. A preparative solvent C having a salting degree range that causes separation of only the target component substance to be separated from the filler 2 among the separated component substances held as a band is introduced. This preparative solvent C is the developing solvent B.
The same phosphate buffer, KCI solution, Na1L solution, etc. are used, but the salt concentration is adjusted to the concentration range at which detachment of the target component substance occurs.

Then, only the target component substance is selectively separated from the packing material 2, flows out from the outlet 4 of the column 1 together with the preparative solvent C, and is fractionated. Other component substances can also be separated in the same manner as described above by introducing into the column 1 the separation solvent B having the salt concentration specified for each substance.

Alternatively, the salt concentration of the preparative solvent C introduced into the column 1 can be adjusted steplessly (Fig. 2) or stepwise (Fig. 3) according to a preset gradient time course plan.
As the salt concentration of the introduced preparative solvent C changes over time, each of the separated component substances retained in the column packing material 2 is sequentially introduced into the packing material 2 while changing the salt concentration over time. It separates from the column and flows out from the outlet 4 of the column 1 together with the preparative solvent C. Therefore, each fractional component substance-containing solvent stream sequentially flowing out from the chain 4 is fractionated and collected for each individual component substance-containing solvent stream portion, or for each component substance-containing solvent stream portion of interest.

(c) Column cleaning, etc. After completing the separation of one, several, or all of the target component substances held in the column packing material 2, they are released into the column packing material. A cleaning operation for the filler 2 is carried out by introducing a solvent having a salt concentration that allows all substances remaining adsorbed to be removed without being absorbed.

Thereafter, by repeating the operations (a) Φ (b) to (c) as one cycle, it is possible to sequentially and fractionally separate the required component substances from the raw material liquid.

The individual solvents containing the desired preparative component substances are concentrated to the required concentration in a concentrator. Alternatively, only the desired preparative component substances can be separated by a drying operation or the like.

As mentioned above, liquid chromatography is effective as a separation and purification method for substances, but in particular, the in-column packing material 2
as hydroxyapatite crystals (usually chemical group 1 &
Ca+o (po, ) b (OH) 2, Hexagonal degree lattice: aAb-120°, a A C= b
A C-90c', 1al= 1bl= 9.24λ
, 1c) = 8.88^ It is a type of calcium phosphate characterized by plate-like or scaly crystals)
When using other fillers such as ion exchange resins, activated alumina, calcium carbonate, etc., it is difficult to separate amounts of substances with minute structural differences, such as molecular weights of 10' to 109 daltons. of biologically relevant polymers (
Proteins such as immunoglobulins, interferons, and enzymes, nucleic acids such as RNA, DNA, and plasmids, and viruses can be separated with sensitivity and precision. It is attracting particular attention as a means of separating and purifying to high purity various useful substances synthesized by recombination, cell fusion, cell mass culture methods, etc.

[Problem that the invention seeks to solve]

In the past, there have been systems in which the separation and purification operations of substances by liquid chromatography as described above are implemented as much as possible. However, these are simply simple processes in which the chromatographic system operation proceeds mechanically according to the initially set program.For example, for some reason, the salt concentration of the solvent actually flowing through the system is acceptable. Regardless of whether the system is out of range, the flow rate of the fluid actually flowing through the system is outside of the allowable range, or an accident has occurred such as air bubbles forming in the system flow path, the system is a purely mechanical process. For this reason, a discrepancy easily occurs between the planned ideal chromatographic process and the chromatographic process that is actually being carried out, resulting in low reliability in terms of preparative separation accuracy and chromatographic data.

In view of the above, the present invention is extremely reliable and can be fully put into practical use as an industrial substance separation device in the biotechnology field, which requires, for example, high-precision target substance separation ability and large-scale processing ability. The purpose is to develop and provide a variety of system equipment.

Structure of the Invention [Means for Solving the Problems] That is, the present invention involves mutually chromatographically separating and developing each component substance in the M solution, which is introduced with a solid filler as a stationary phase packed inside. a chromatographic separation column system consisting of one column or a plurality of columns; a raw material solution supply device for introducing the raw material solution into the columns of the column system; A solvent supply device that introduces the salt concentration of the solvent solution while changing it steplessly or stepwise according to the standard gradient time course setting, and each fraction component substance that sequentially flows out from the column of the column system. A fraction collator that collects the contained solvent stream separately for each component substance-containing solvent liquid part or for the desired component substance-containing solvent liquid part, and a part of the solvent flow that enters the column of the column system from the solvent supply device. , or a salt concentration measuring device that receives a portion of the solvent flowing out from inside the column of the column system as a sample solvent and measures the state of change in the salt concentration of the solvent over time; A component substance separation state measuring/monitoring device that receives one part as a sample solvent and measures the separation state of each component substance fractionated from the column sequentially over time.

A solvent flow measuring device that measures the flow rate of a solvent stream flowing into a column system, the flow rate of a solvent stream flowing out from a column of a column system, or both over time; and a solvent salt output from the salt concentration measuring device. The measured value of the concentration change over time is compared with the standard salt concentration change function value over time, and the comparison information is fed back to the salt concentration change over time control means to control the salt content of the solvent introduced from the solvent supply device into the column of the column system. A salt concentration automatic adjustment system for appropriately correcting the concentration and a measured value of the change in solvent flow rate over time outputted from the solvent flow i measurement device are compared with a reference flow rate value, and the comparison information is fed back to the flow rate control means. A system control device that includes an automatic solvent flow rate adjustment system that appropriately adjusts the flow rate of the solvent introduced from the solvent supply device into the column of the column system, and that coordinately controls each of the above-mentioned component devices that make up the system according to predetermined operating conditions. and.

The subject matter is a system for separating and purifying substances by liquid chromatography, which is characterized by:

[For production]

A major feature of the system device of the present invention is that the salt concentration and flow rate changes over time of the solvent actually flowing upstream into the system device are automatically measured and compared with respective appropriate values.
A control system that feeds back such comparative information to the salt concentration temporal change control means and the flow rate control means, and automatically controls the salt concentration and flow rate of the solvent actually upstream to the system device so that they always maintain appropriate setting conditions. As a result, even if a setting condition deviation occurs for some reason in the chromatography process currently in progress, the deviation in setting conditions is automatically corrected immediately, so that the chromatography process is maintained under ideal setting conditions from beginning to end. is executed. In other words, an extremely reliable system device is constructed.

〔Example〕

FIG. 1 is a block diagram showing the configuration of an embodiment of the apparatus according to the present invention.

10 is one column or a plurality of columns packed with a solid packing material such as hydroxyapatite crystal as a stationary phase and mutually chromatographically separates and develops each component substance in the introduced raw material liquid. This is a chromatographic separation column system.

11 is a raw material liquid supply device, which is a storage tank 1 for raw material liquid A;
2, a raw material liquid extraction pump 13, a raw material liquid injector 14 for injecting a predetermined amount of raw material liquid into the piping 15 on the fluid inlet side of the column I, and the like.

Reference numeral 16 denotes a solvent supply device, which includes a storage supply unit 17 for a solvent prepared with a low salt concentration (low concentration solvent), a storage supply unit 21 for a solvent prepared with a high salt concentration (high concentration solvent), and a storage supply unit 21 for supplying both. Confluence mixing chamber 2 of low concentration solvent and high concentration solvent from
It consists of 5.

The low-concentration solvent storage and supply unit 17 includes a plurality of tanks 18.
・18□...18n, the tank selection switching cock device 19, and the cock'! It consists of a pump 20 and the like that introduces the low concentration solvent in the tank selected by the l device 19 into the mixing chamber 25.

Also, the high concentration solvent storage supply section 21 also contains phosphate buffer @K.
A plurality of tanks 22 each storing various solvents such as CI solution and Mail solution prepared with high salt concentration.
222, .

The solvent in the solvent mixing chamber 25 is supplied to the column system lO through the pipe 15.
is introduced into column 1 of. 26 is a solvent mixing chamber 25;
This is a pressure gauge provided in the piping section between the raw material injector 14 and the raw material injector 14.

27 is piping 2 on the fluid outlet side of column 1 of column system 10
8 is an outflow solvent flow i measurement device, and 29 is a fraction collator (Fraction Co1) installed next to it.
1ector) and 30 are concentrators that distribute and condense the respective required component substance-containing solvent liquid portions that have been fractionally collected by the fraction collator.

31 is a branch pipe for collecting a small amount of sample solvent extending from the flow rate measurement device 27; 32, 33, and 34 are bombs for sending sample solvent arranged in series in the branch pipe;
This is a component substance separation state measurement/monitoring device and a salt concentration measuring device.

Reference numeral 35 denotes a system control device (control circuit section). This control device M35 coordinately controls each component device making up the system according to predetermined operating conditions set in advance by the console section 40. Specifically, the raw material liquid supply device M1
1 pump 13, cock equipment M of low concentration solvent supply device 17
19 and pump 20, cock device 23 and pump 24 of high concentration solvent supply device 21, fraction collator 29,
The fIi condenser 130, the sample solvent feed pump 32, etc. are linked and controlled. All pumps in the system are non-pulsating pumps.

In addition, the above control device W135 is a solvent flow measuring device F.
a27, component substance separation state measurement-monitor device 33, fJ
Upon receiving the chromatography rotation progress status information outputted from each of the i concentration measuring devices 34, it performs predetermined system automatic correction control, outputs/displays required data, and issues warnings.

The operation will be explained below.

(2) Setting predetermined operating conditions on the control device 35;

Turn on the system.

■ Solvent 61C is supplied to column 1 of the column system 10 from the solvent supply device 16, and the salt concentration is adjusted steplessly (w42 figure) or stepwise (ti43 figure) according to the preset gradient time course setting. It is introduced continuously while automatically controlling the change over time. Specifically, the automatic time-dependent change control of the salt concentration of the solvent C introduced into the column 1 from the device 16 is performed as follows.

That is, when the system is turned on, the cock in the cock device 19 corresponding to the type of solvent tank (for example, tank 181 storing a low concentration phosphate buffer solution) selected in advance for the low concentration solvent supply section 17 is activated. Control device 35
It is held open by On the other hand, high concentration solvent supply section 1
8, the cock in the cock device 23 corresponding to the type of solvent tank selected and designated in advance (for example, the tank 221 storing high concentration phosphate buffer) is held open by the control device 2t35.

Next, the pump 20 is activated and the low concentration solvent Ct is introduced into the mixing chamber 25. Also, the pump 24 is activated and the high concentration solvent Co is introduced into the mixing chamber 25 as well. The introduction of both solvents CL and CH into the mixing chamber 25 is controlled by the pump 20 and the control device 35 of the same 24, with the low concentration solvent CL being introduced in large quantities at first, and thereafter in accordance with a predetermined decreasing function over time. Contrary to the above for the highly concentrated solvent CH, the amount introduced is zero or very small at first, and then the amount introduced is increased according to a predetermined increasing function over time, and the amount is increased in the mixing chamber. The total amount of both soluble 6ICL and CH introduced into 25 is controlled to maintain a substantially constant relationship over time.

As a result, the solvent C is introduced from the mixing chamber 16 into the column 1 while the salt concentration thereof changes steplessly or stepwise in accordance with a preset gradient setting over time.

■Since the introduction of solvent C into column 1 has started 1
．． At this point, raw material liquid supply? The operation of the pump 13 of the device 11 is controlled by the control device 35 to pump a predetermined amount of raw material m.
A is injected into the column l from the mixing chamber 25 by the injector 14.
The raw material liquid A enters the column 1 together with the solvent C.

(2) As a result, each component substance in the raw material liquid A is chromatographically separated and developed in the column 1 between the raw material introduction time 1 and the time t2.

■Solvent C continues to be supplied into column L, and the salt concentration increases over time, until the salt concentration reaches its maximum value (
Until the time point t3 when the concentration of high concentration solvent CH is reached, each developing substance during chromatographic separation retained in the column packing material as the salt concentration changes over time.
* are sequentially separated from the filler and flow out into the pipe 28 together with the solvent C.

(ΦThe effluent solvent is then separated and collected by the fraction collator 29 for each component-containing solvent liquid portion that sequentially flows out, or for the desired useful component-substance-containing solvent portion.

The solvent portion containing unnecessary component substances is drained 36.

(2) Next, the separately collected solvent solutions containing the respective useful component substances are introduced into the concentrator 30 and taken out as concentrated liquids.

■On the other hand, the actual flow rate of the solvent flowing out from the force ram 1 into the pipe 28 is measured over time by the flow rate measuring device 27, and the measured value over time is input to the control device 35, and it is determined based on the preset standard. The pump 20 of the solvent supply device 16 and/or Same 2
The operating states of 4 are automatically controlled in relation to each other.

(2) Also, a small portion of the solvent flow flowing out from the column 1 to the pipe 28 is branched off from the branch pipe 31 extending from the flow i measuring device 27 as a sample solvent. The sample solvent is continuously sent by a pump 32 to a component separation state measurement/monitoring device 33 and passes through the device. During the passing process, the device 33 measures and monitors the state of each component substance sequentially separated from the column 1 over time (second
The dashed line in the figure is the monitor chromatogram), and the information is input to the control device 35. This measurement and monitoring of the state of sequential separation of each component substance over time is performed with high accuracy, for example, by a method of measuring changes over time in the ultraviolet absorption characteristics of the sample solvent.

[Co., Ltd.] The sample solvent that has passed through the device 33 enters the next salt concentration measuring device 34, and the actual change over time in the salt concentration of the solvent C flowing through the system is measured. The sample solvent after measurement is drained 37.

Changes in the salt concentration of solvent C over time can be detected, for example, by a method that measures changes in the refractive index of solvent C over time, a method that measures changes in electrical conductivity over time, etc., but the former refractive index measurement method is better. Compared to the latter method, this method is more practical because it can detect linearly and accurately without electrolytic phenomena.

And the measured values over time are the control device! 135, the measured value over time is compared with a preset standard salt concentration time change function value (planned gradient time course setting), and the comparison information is fed back to the salt concentration control circuit. The operating states of the solvent supply device 16 and the pump 20 and/or 24 are automatically controlled in relation to each other so that the change over time in the salt concentration of the solvent introduced into the column I is maintained at the predetermined course setting. .

0 During the period from time t3 when the salt concentration of solvent C introduced into column 1 reaches the maximum value to predetermined time t4, column 1
Subsequently, solvent C is introduced into the chamber at the maximum salt concentration.

During this time, all the adsorbed components remaining in the packing material in the column are separated and drained from the collator 29 as a liquid 36, thereby cleaning the inside of the column 1.

From O onwards, the same cycle is automatically repeated as one preparative operation cycle from ■ to ◎ above, and the target component substance is extracted with high precision from the raw material liquid that is intermittently introduced into the column from the raw material liquid supply device 11. It is continuously separated.

Note that the salt concentration of the solvent is measured using pipe 1 on the solvent introduction side into the column.
Alternatively, a sample solvent may be taken out from the 5 side and the salt concentration of the sample solvent may be measured.

C1 Effects of the Invention As shown in C, the system device of the present invention is capable of immediately and automatically correcting the setting condition deviation even if a setting condition deviation occurs for some reason in the ongoing chromatography process. The process is very reliable, carried out practically according to ideal set conditions throughout, and the intended purpose is well achieved, making it extremely effective and suitable as a preparative system for useful substances, for example in the field of biotechnology. be.

[Brief explanation of drawings]

Fig. 1 is a block diagram showing the configuration of the system device of one embodiment, Fig. 2 is an example of a graph for setting a stepless change in solvent salt concentration over time, Fig. 3 is an example of a graph for setting a stepwise change over time, and Fig. The figure is a longitudinal sectional view showing an example of a column configuration. 1 is a column, 11 is a raw material liquid supply device, 16 is a solvent supply device, 29 is a fraction collator, 30 is a concentrator, 3
5 is a system control device.

Claims (4)

[Claims] (1) A solid packing material is packed inside as a stationary phase, and each component substance in the introduced raw material liquid is chromatographically separated from each other.
A chromatographic separation column system consisting of one column or a plurality of columns to be developed; a raw material solution supply device for introducing a raw material solution into the columns of the column system; A solvent supply device that introduces the salt concentration of the solvent solution while changing it steplessly or stepwise according to the standard gradient time course setting, and each fraction component substance that sequentially flows out from the column of the column system. A fraction collator that collects the contained solvent stream separately for each component substance-containing solvent liquid part or for the desired component substance-containing solvent liquid part, and a part of the solvent flow that enters the column of the column system from the solvent supply device. , or a salt concentration measuring device that receives a portion of the solvent flowing out from inside the column of the column system as a sample solvent and measures the state of change in the salt concentration of the solvent over time; A component substance separation state measurement/monitoring device for measuring the separation state of each component substance collected sequentially over time from within the column by receiving one part as a sample solvent, and a flow rate of the solvent flow flowing into the column system, or A solvent flow rate measuring device that measures the flow rate of a solvent flow flowing out from a column of a column system, or both over time, and a measured value of a change in solvent salt concentration over time output from the salt concentration measuring device and a reference salt concentration. A salt concentration automatic adjustment system that compares the temporal change function value and feeds back the comparison information to the salt concentration temporal change control means to appropriately correct the salt concentration of the solvent introduced from the solvent supply device into the column of the column system. , and compares the measured value of the change in solvent flow rate over time outputted from the solvent flow rate measurement device with a reference flow rate value, and feeds back the comparison information to the flow rate control means and introduces it from the solvent supply device into the column of the column system. a system control device that includes a solvent flow rate automatic adjustment system that appropriately corrects the solvent flow rate that is applied, and that coordinately controls each of the above-mentioned component devices constituting the system according to predetermined operating conditions. Separation and purification system equipment for substances using graphics. (2) The system device according to claim (1), wherein the salt concentration measuring device is a device that detects a change in salt concentration over time by measuring a change in the refractive index of a sample solvent over time. . (3) The component substance separation state measurement-monitor device is a device of a type that measures changes over time in the ultraviolet absorption characteristics of the sample solvent and detects the separation state of each preparative component substance over time. A system device according to claim (1). (4) The system device according to claim (1), wherein hydroxyapatite crystals are used as the solid filler in the column.