JPS61270658A - Separating and refining device - Google Patents

Abstract

PURPOSE:To easily reproduce an eluate and monitor composition by reproducing the eluate by film separation and monitoring the composition of the eluate by detecting and controlling it with the eluation pattern of a target component. CONSTITUTION:Mixed liquid which contains a material to be refined is added at the upper part of a column 5 and the eluate is sent from an eluate storage tank 1 to a line mixer 3 through a valve 2 by a high pressure pump 4, mixed, and fed to the column 5. A difference in moving speed is generated in the column 5 owing to the material and materials which are eluated are detected by a detector 6 in order of eluation; and a valve 7 is opened on the basis of previously inputted information to gather fractions containing only the target material to be refined by a collector 8. Compatible materials are removed by a film module 9 from an eluate which contains materials other than the target material and the eluate is adjusted to initial eluate composition and sent to the column 5 again by the pump 4 to circulate repeatedly. The eluation time of the target material from the column is inputted to a computer previously and if a time difference is generated, a valve 2 is opened to control the composition of the eluate automatically.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an apparatus for separating and purifying mixtures on an industrial scale, and more particularly to a liquid chromatography apparatus having an eluent regeneration function.

Liquid chromatography is a technology originally developed as a laboratory-scale analysis device, but it can also be obtained by using biotechnology such as genetic recombination and cell fusion. It has come to be used to separate and purify substances with high purity and in a short time on an industrial scale. This method involves filling a pressure-resistant column with silica gel, ODS, and porous polymer gel, adding the mixture to the top of the column, and then extruding it from the column with /8 syneresis to remove the target component from the mixture by +1iu-t. This is the way to do it.

When applying liquid chroma 1 to graphie as an industrial separation and purification technology, each time the column size increases, the processing amount increases, and the amount of water per month also increases accordingly. As 1/& (31), depending on the gel to be filled and the target substance, acetonitrile solutions are often used.

The invention solves the problems, especially as a release agent. /'When using J medium (31
Therefore, it is necessary to recycle the Y1 organic solvent from a simultaneous point of view. Therefore, we collect and purify the organic solvent by distillation,
A method of 1fi Ikawa is being used, but because it is expensive, the range of practical application is often limited, although there are 14 points in liquid lithography such as separation, purification, purity, or high speed.

On the other hand, when the /8 syneresis solution is used repeatedly, if the composition of the eluent changes, the -1 substance will be eluted from the column at the 11th time (retention time) due to a shift of η". , the separation degree of adjacent peaks decreases, and the purity of the target substance decreases accordingly.Therefore, monitoring of the eluent composition becomes an important issue.Therefore, the eluent should be recycled. In some cases, a monitoring device (+1V) is required, and a large amount of equipment is required.

The present invention (,31, solves the drawbacks of the above-mentioned prior art/l!i
(-,, cold cloth 1 [11 cows and composition of the liquid to be easily monitored in CY (to make a small V2 as much as possible to 1 Oj-J'i]), and the purpose is 4) .

Means for Solving Problems) The present invention re-re/L1'/"6i'ill/& by membrane separation, and monitors the eluent composition by detecting the acid component elution pattern, 31- to control;
The above-mentioned problems have been resolved by IW.

Therefore, the separation and purification equipment W U91, RI 1-17 according to the present invention
1 Graph E carafe inflow '1 side elution lfk storage tank,
Connect the eluent flow line mixer to the +IC 11 from II history module before the line mixer.
+ 1/3 of the circulation of i'a Oka 11 liquid is merged with the mouth, ri LJ
71 A component detector and a recording of the detection pattern are installed on the outlet side of the graphics column, and the fractions are passed through the outflow pipe 4;Zi;I valve from the component detector: Lucter and Rise Schiff.

The valve is opened and closed based on the information entered into the computer in advance, allowing the eluate showing the target substance to flow out to the fraction collector, and the other /8 separate fluid flowing out to the crotch module. It is characterized by

Non-inventive number J, the liquid used as a dissolution liquid is alcohol,
It is a low-molecular substance such as acetonitrile, and conversely, a mixture is
Since liquid chromatography is used in the final stage of industrial separation and purification processes, there are few impurities, and the target substances are often high molecular substances such as sugars and proteins. We focused on the fact that the retention time of the target substance and the retention time of the target substance were already monitored separately using an ultraviolet absorption meter, differential refraction 11, etc., and in order to make effective use of this feature, we regenerated the eluent through membrane separation and then detected the target substance. The system is designed to detect the retention time of 11i and keep the solution silk composition constant based on the change.

Function: In the fraction III purification device according to the present invention, the detector provided on the outlet side of the chromatography column is
The component pattern of the fraction flowing out from the column is detected, and the detection results are output to the 21st column. This, :l
Based on the information entered in advance, the computer
Open and close the valve to allow the fraction to flow into the fraction collector or crotch module, or by external force,
Open and close the inlet valve for the new synergic liquid.

The membrane module removes coexisting substances from the fraction flowing out from the membrane and generates an eluent internally.

The regenerated eluent is mixed with fresh eluent according to its composition by a line mixer, and is always passed through the column as an eluent with a constant composition.

Example Next, the present invention will be explained in detail based on the drawings.

FIG. 1 is a flow sheet of a separation viscodissection device showing one embodiment of the present invention. When carrying out separation and purification using this apparatus, first, a liquid mixture containing the substance to be purified is added to a portion of Karano-5. The eluent is sent from the liquid storage tank 1 through the valve 2 to the line mixer 3 using the high pressure pump 4.
After mixing thoroughly, the solution is sent to the column. In the column 5, there are differences in movement speed depending on the substance, and the substances eluted from the column are detected by the detector [i], and the valve 7 is opened based on the input information +U, and the purification is carried out. The fraction containing the six-dimensional substance obtained is collected in a fraction collector 8.

The detection pattern 4J detected by the detector 6 is recorded in the record 10.

Le as a factor determining the severity of substance movement in humans
t, the type of gel packed in the column, the composition of the eluent, and the physicochemical characteristics of each component in the mixture, which are determined depending on the characteristics of the substance to be purified.

The eluent containing substances other than the target substance has coexisting substances removed in the membrane module 9, and the initial /8 separation l&i11
The mixture is then sent to the column again using the high-pressure pump 4 and the circulation is repeated.It is thought that repeating the circulation will change the liquid composition. 1) Enter the time for the target substance to elute from the column (retention time) into the 22-meter monitor in advance, and if there is a discrepancy in the retention time, open valve 2 and elute. The composition of the liquid is 1%.

Based on the conditions that are often used on an industrial scale (gel: 01S, eluent: a mixture of water and methanol), the following conditions are shown in Figure 2. Zl-J
It is known that the retention time of the target components changes depending on the degree of methanol, as shown in FIG. Therefore, by understanding this relationship in advance according to the target component and inputting the allowable range of change in retention time (set by taking into account the retention time of other substances to be removed) into the computer, it is possible to adjust the valve accordingly. Allow automatic control.

In addition, as a membrane module, 1 volume M? Since organic solvents such as alcohol and acetonyl are often used at night, solvent-resistant membrane modules such as polyamide-based membrane modules are preferred.

Example 1 In the apparatus shown in FIG. 1, an aqueous GPC gel (
exclusion limit molecular weight 300,000), diameter 5 cm, height 60
Packed into a 50mM phosphorus buffered column (p
1 volume of water containing H7,5') and 0.2 M NaCl was used as the first liquid in the final container, and was flowed at a flow rate of 50 m1/min.
Mixture of ovalbumin and bovine serum albumin for 1 minute & 1
As a result of 1 purification, efficient separation and purification was achieved.

Invention - Φ - Effect According to the present invention, the membrane module which is an energy saving device? G! The ttII fluid can be regenerated and reused. In addition, computers for detecting eluted substances from columns and opening and closing valves for fractions have been installed in the past, and these can be effectively combined without adding new equipment. With facilities,
Able to perform and move NM in minutes efficiently.

[Brief explanation of the drawing]

Figure 1 (J, of a separation and purification device showing one embodiment of the present invention) [Cosheet, Figure 2 is a schematic diagram of the peak pattern of eluted components, and Figure 3 I♀1ba/8 synergic composition is the elution time of the component FIG.

Claims (2)

[Claims] (1) In an apparatus for separating and purifying mixtures by liquid chromatography, an eluent storage tank, an eluent inlet valve, and a line mixer are connected to the inlet side of the chromatography column, and the regenerated eluent from the membrane module is connected before the line mixer. The liquid circulation paths are merged, a component detector and a detection pattern recorder are installed on the outflow side of the chromatography column, and a fraction collector and membrane module are connected to the outflow pipe from the component detector via a valve. , the separation and purification device is characterized in that the valve is opened and closed based on information input into a computer in advance to allow the eluate showing the peak of the target substance to flow out to a fraction collector, and the other eluent to flow out to the membrane module. . (2) The separation and purification apparatus according to claim 1, wherein the retention time of the target substance is input into a computer, and the eluent inflow valve is opened and closed according to changes in the retention time.