JPS6360863B2 - - Google Patents

Description

Detailed Description of the Invention [Industrial Application Field] The present invention involves sampling a reaction solution at regular time intervals in order to track the enzymatic reaction progressing in a reaction tank, and analyzing the reaction solution using liquid chromatography. The present invention relates to a dilution sampling method for diluting a sampled reaction solution and injecting the diluted sample into the liquid chromatography apparatus in order to analyze components.

[Conventional technology]

In a chemical reaction such as an enzymatic reaction, in order to examine the progress of the reaction in detail, it is necessary to analyze the concentrations of multiple components in the reaction solution at regular intervals. Therefore, according to the conventional method, a fixed amount of the reaction solution is collected into a test tube using a pipette at fixed intervals, and a diluted solution, quenching agent, protein removal agent, etc. are added to the test tube and mixed thoroughly. After that, the supernatant after removing the precipitate is analyzed by liquid chromatography. The mixture is analyzed by liquid chromatography. The liquid mixture is injected into the liquid chromatograph using a microsyringe or an autosampler for liquid chromatography. However, when using any of the above injection methods, the sampling and pretreatment of the reaction solution depend on manual operations, which tends to introduce uncertainties such as the timing of sampling the reaction solution and the dilution rate. There are problems such as the high possibility that foreign substances such as microorganisms may be mixed into the reaction tank during collection, and automation is difficult.

[Problem that the invention seeks to solve]

SUMMARY OF THE INVENTION In order to eliminate the above-mentioned drawbacks, the present invention provides a dilution sampling method that makes it possible to automatically sample a reaction solution from a reaction tank and inject the diluted sample into a liquid chromatograph apparatus.

[Means for solving problems]

The dilution sampling method according to the present invention includes a plurality of sampling valves, a plurality of flow path switching valves,
Equipped with a sealed flow path consisting of a stirring container, liquid pump, line filter, piping, etc., a fixed amount of reaction liquid sampled by a sampling valve connected to the reaction tank is uniformly distributed with a diluted solution in the closed flow path. After diluting and injecting a fixed amount of the diluted sample into the liquid chromatograph apparatus using a sampling valve connected to the liquid chromatograph apparatus, after washing by combining the switching of the plurality of flow path switching valves and reversing the liquid feeding pump, The present invention relates to a diluted sampling method characterized by replacing the solution in a closed channel with a fresh diluted solution.

〔Example〕

DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be further explained below with reference to the drawings.

In FIG. 1, a reaction tank A, a liquid feeding pump P ₁ ,
The sampling valve S ₁ is connected by piping,
A reaction solution is constantly circulated within the piping. When the sampling valve S ₁ is switched, the reaction solution held in the sample loop L ₁ is sampled into the closed channel T, and the reaction solution is transferred to the sampling valve S ₁ ,
Flow path switching valve V ₂ , stirring container V, liquid sending pump R ₁ ,
R ₂ , flow path switching valve V ₁ , line filter F,
While passing through the sampling valve S ₃ etc. and circulating in the closed channel T from 1 to 10 times, the reaction solution is uniformly diluted with the diluted solution that previously filled the stirring vessel V and the closed channel T. be done. The line filter F is inserted between the flow path switching valve V ₁ and the sampling valve S ₃ for the purpose of removing insoluble fixed substances present in the closed flow path T. The uniformly diluted sample solution is transferred to the flow path of the liquid chromatograph device _H1 by switching the sampling valve _S3 and holding the diluted sample solution in the sample loop _L3 , and then returning the sampling valve _S3 again. injected into.

After injecting the diluted sample solution into the liquid chromatograph apparatus _H1 , the solution in the closed channel T is replaced with a fresh diluted solution as follows. That is, the flow path switching valve V ₂ is switched, the flow path switching valve V ₁ is set to position 2, the solution in the stirring container V is discarded from the waste liquid port W ₁ , and then the flow path switching valve V ₁ is set to position 2. At the same time, the liquid sending pumps R ₁ and R ₂ are reversed, and the stirring container V is filled with the cleaning liquid from the cleaning solution container B ₂ while cleaning the closed channel T. After repeating the above-mentioned waste liquid operation and filling operation alternately from one to five times, return the sampling valve _S1 to the standard position, discard the cleaning solution in the stirring container V from the waste liquid port _W1 , and then At the same time, the flow path switching valve V ₁ is set to position 1, and at the same time, the liquid transfer pumps R ₁ and R _{2 are}
is inverted, and the stirring container V is filled with the diluted solution containing the quenching agent, protein removal agent, etc. from the diluted solution container _B1 . Return the flow path switching valve V ₂ to the standard position,
When the liquid sending pumps R ₁ and R ₂ are rotated in the normal direction, preparations for receiving the next sample solution from the reversing tank are completed.
In this embodiment, a control device with a built-in clock, such as a computer or a sequence controller, outputs an electrical signal corresponding to a predetermined operation at a predetermined time according to the operation procedure, and
A plurality of valves and liquid pumps each having a drive unit adapted to the electric signal are operated.

FIG. 2 shows the embodiment shown in FIG. 1, and further includes a sampling valve S ₂ , a liquid feed pump P ₂ , a standard sample solution B ₃ ,
Sampling valve S ₄ , liquid chromatography device
In the case of a sample in which _H2 has been added and the rate of change in the concentration of the reaction solution components exceeds the analysis speed of the liquid chromatograph device _H1 , the sample should be injected alternately into two liquid chromatograph devices. This is a dilution sampling device for parallel analysis.
In addition, the same sample can be processed using two liquid chromatography devices.
It is possible to analyze H ₁ and H ₂ under separate conditions to improve resolution, detection dynamics, number of components that can be analyzed, etc. It is thus possible to further maintain a liquid chromatography device for multiple purposes. The elution position and elution intensity of the analytical components in the liquid chromatogram are determined by each liquid chromatography device H ₁ ,
Because H ₂ differs slightly, the same standard sample was analyzed in advance with multiple liquid chromatography devices to compensate for the differences in characteristics between each device.
It is preferable to obtain a correction coefficient in advance, and in this embodiment, a sampling valve for a standard sample is provided.

The diluted solution is selected as appropriate depending on the target sample, but usually a uniform or slurry reaction solution is uniformly dissolved in water, buffer solution, organic solvent, quencher solution, protein remover solution, etc. Select and use a solution that is similar to the eluent of liquid chromatography and does not adversely affect the chromatogram. The washing solution is usually water, a buffer solution, an organic solvent, etc., and is preferably a solution that mixes freely with the reaction solution and diluted solution and does not affect the enzymatic reaction proceeding in the reaction tank A at all. The standard sample solution preferably contains the same substrate and product as the reaction solution, but without the enzyme. sampling valve
The sampling loop capacitance of S ₁ and S ₂ is 10μ or more1
ml or less, the sampling loop capacity of the sampling valves S ₃ and S ₄ is preferably from 10 μm to 100 μm, and the volume of the stirring vessel V is preferably from 0.5 ml to 10 ml. The dilution ratio is determined from the sampling loop volume and the volume of the stirring vessel, and is appropriately selected depending on the component concentration of the reaction solution and the sensitivity of the detector. Liquid pump R,
P ₁ and P ₂ may have a liquid feeding amount of 10 ml or more and 1000 ml or less per minute. Liquid chromatography device H ₁ ,
H ₂ is a conventional device equipped with a liquid pump, a pulse damper, a pressure gauge, a separation column, a chromatographic detector, a recorder, etc., and analysis is performed in gel filtration mode, distribution adsorption mode, etc. The eluent for liquid chromatography is appropriately selected depending on the sample to be analyzed, and preferably includes water, buffer solution, methanol, acetone, acetonitrile, and the like.

As a specific example, when measuring a peptide decomposition synthesis reaction using a protease, the peptide is analyzed by reverse phase chromatography in a water-acetonitrile mixed solvent system, so both the diluted solution and the eluent are in a water-acetonitrile mixed solvent. The cleaning solution is preferably a water-acetonitrile mixed solvent or pure water.

〔Effect of the invention〕

If the dilution sampling method according to the present invention is used, the dilution sampling device has no dead volume, so that only a trace amount of fresh reaction solution can be sampled from the reaction tank without loss. This method has advantages such as being able to dilute the sample solution and stop the enzyme activity at the same time. Furthermore, even when the reaction solution is in the form of a slurry, it is possible to uniformly dissolve the slurry by appropriately selecting a diluted solution; for example, a mixed solvent of water and acetonitrile is used to dissolve peptides.

[Brief explanation of drawings]

FIGS. 1 and 2 are block diagrams showing the configuration of a diluted sampling method according to each embodiment of the present invention. S ₁ , S ₂ , S ₃ , S ₄ ...sampling valve, L ₁ ,
L ₂ , L ₃ , L ₄ ... sampling loop, V ₁ , V ₂ ... flow path switching valve, P ₁ , P ₂ ... liquid pump, R ₁ , R ₂ ...
Liquid feed pump, F...line filter, A...reaction tank, V...stirring container, M...magnetic stirrer,
_B1 ...Diluted solution container, _B2 ...Washing solution container, _B3 ...Standard sample, T...Sealed channel, _H1 , _H2 ...Liquid chromatograph device, _W1 , _W2 ...Waste liquid port.

Claims (1)

[Claims] 1. In a method of diluting and sampling reaction solution components in an enzyme reaction, in order to maintain a constant amount of reaction solution from a reaction tank, the reaction solution is constantly circulated through a sampling loop of a sampling valve, and the sampling valve is closed. By switching, the reaction liquid held in the sampling loop is uniformly diluted with a diluted solution in a closed flow path consisting of a line filter, a stirrer, multiple flow path switching valves, liquid delivery pump, etc., and the liquid chromatography device and switching the plurality of switching valves to connect a flow path from a cleaning solution container to a point in the closed flow path, and connect a waste liquid inlet to a point in the closed flow path, thereby switching the flow path from the cleaning solution container to a point in the closed flow path. Washing the inside of the channel with a cleaning liquid, then switching the plurality of switching valves to connect the flow channel from the diluted solution container to one point of the sealed channel, and connect the waste liquid port to one point of the sealed channel, A diluted sampling method characterized in that the inside of the sealed channel is thereby replaced with a diluted solution. 2. The diluted sampling method according to claim 1, wherein after performing the step, the steps are further repeated. 3. Equipped with a plurality of valve switching drive units each operated by switching a multi-point external output signal of a control device and a liquid feeding pump having a function of reversing the liquid feeding direction operated by switching the signal, The diluted sampling method according to claim 1 or 2, wherein sequential control is possible by an external output signal of the control device. 4 Equipped with a sealed flow path consisting of a sampling valve connected to a reaction tank, a sampling valve connected to a liquid chromatograph device, multiple flow path switching valves, a stirring container, a liquid feeding pump, a line filter, piping, etc. , by switching the sampling valve connected to the reaction tank, the portion held in the sampling loop at the moment of switching is sampled from the reaction solution that is constantly circulated between the reaction tank and the sampling valve by a liquid feed pump. After uniformly diluting the sample with a diluted solution in a sealed channel, a certain amount of the diluted sample solution is supplied to the liquid chromatograph by switching the sampling valve connected to the liquid chromatograph, and then By switching multiple switching valves,
A flow path from a cleaning solution container is connected to a point in the closed flow path, a waste liquid port is connected to a point in the closed flow path, thereby cleaning the inside of the closed flow path with a cleaning solution, and then the plurality of Switch the switching valve to connect the flow path from the diluted solution container to one point in the sealed flow path, connect the waste liquid port to one point in the closed flow path, and thereby replace the inside of the closed flow path with the diluted solution. A diluted sampling method characterized by: