JPS63265166A - Rotary type automatic thin layer chromatography apparatus - Google Patents

Abstract

PURPOSE:To achieve quick separation and to improve resolution, by detecting the peak separating state of each component of a sample on a separating means of a rotary type automatic thin layer chromatography apparatus, and controlling the rotation of the separating means in correspondence with the result. CONSTITUTION:A separating part S has a separating means F, which is formed by providing a solid phase for a thin layer chromatography on a disc shaped substrate, and a motor M serving as a rotating and driving means of the separating means F. The temperature of the separating part S is regulated with a temperature regulator 1. A detector D comprises a driving part 3, a peak detecting part 4 and a position detecting part 2. The peak detecting part has a light source 5 and a light receiving part 6. Light is projected on the separating means F, and the reflected light corresponding to each component on the separating means is received in the light receiving part. A required peak signal is transmitted in correspondence with the reflected light. The signal which is detected in the peak detecting part is sent to an operation control part C. Operation is performed from the peak separation signal. The rotation of the motor M in the separating part is controlled according to the result of the operation.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a rotary automatic thin layer chromatograph.

According to the present invention, each component in a sample can be efficiently separated in large quantities in a very short time, so it can be used not only for quantitative and qualitative analysis, but also for large-scale separation and purification of specific substances. Therefore, it is particularly useful for industrial production or industrial separation of specific substances such as physiologically active substances.

[Conventional technology]

Chromatography, particularly liquid chromatography, has been widely used for the analysis and separation of physiologically active substances, chemicals, and various other substances.

However, liquid chromatography is carried out using a device consisting of a separation section consisting of a column and a liquid pump, and a detection section, but this device not only requires a liquid pump, but also requires It is necessary to send the liquid at high pressure to the column, and in addition, the development method using conventional preparative columns has the following disadvantages: ■ It takes a long time to develop. ■ It is difficult to perform large-scale separation. ■ It takes time to develop conditions. be.

Therefore, as an improved method, a thin chromatographic layer is formed on a disk-shaped substrate, which is then rotated, and the solvent is spread radially from the center using centrifugal force to separate and fractionate the target substance. A method is proposed. However, although this method alone increases the development speed, the separation performance often deteriorates, and it can only be used on a laboratory scale.

As described above, the conventional method of using a rotary automatic thin layer chromatograph as it is cannot be used for industrial large-scale processing because delicate processing operations cannot be performed.

The present invention organically adds to a rotary automatic thin layer chromatograph a detection section that detects the separation state of each component and generates a required signal, and a means for controlling the rotation speed of the separation means in response to the signal. This makes it possible for the first time to not only completely separate each component in a short time, but also to separate them in large quantities on an industrial scale. This technology succeeded in raising the level of industrial production, in which the target substance is industrially separated and collected in addition to analysis, but such technology is completely unknown and new.

Furthermore, regarding the technical idea of automation and industrialization of such a rotary thin layer chromatograph, which automatically performs separation while delicately controlling the rotational speed according to each component, as in the present invention, Conventionally, there has been no such suggestion, and the technical problem to be solved by the present invention is already new.

[Problem that the invention seeks to solve]

Such a rotary thin layer chromatograph has sufficient separation power in special cases where the number of components of the substance to be separated is small and the Rf values of each component are at regular intervals, but in reality, One substance contains various impurity components, and the difference in Rf value of each component spot is close,
They are far apart and diverse.

In order to solve this problem, the conventional method used was to delicately control the polarity of the developing solvent, but since separation performance varied depending on the components, several trials were required to obtain better separation conditions. You have to repeat your mistakes. That is, there is a problem that it takes a very long time to set the conditions.

In addition, a large amount of sample is required for condition determination,
As a result, there is a problem in experimental efficiency, such as when the amount of sample is small, valuable samples are wasted in order to determine the optimal conditions.

[Means for solving problems]

The present invention has been made in view of the current state of technology, and has the inherent advantages of a rotary automatic thin layer chromatograph in that it does not require a liquid pump and can quickly separate components. This was done in order to efficiently and completely separate each component by increasing the low component separation ability, which is a structural drawback, while making the most of this.

At the same time, the present invention was made for the purpose of completely automating the above processing and industrializing it so that it can be processed in large quantities. Therefore, the present invention has the purpose of not only simply analyzing but also separating and collecting target components.

As a result of various studies to solve the above problems and achieve the objectives, we found that it is necessary to: ■ be able to detect the separation state in real time, and ■ have a mechanism to control the deployment state based on the state of ■. We came to the conclusion that there is a system for thin-layer chromatography, and as a result of intensive consideration of ways to concretely achieve each of these requirements, we formed a stationary phase for thin-layer chromatography on a disk-shaped substrate and rotated each component in the sample. In a disk-shaped thin layer chromatograph that develops radially sequentially from the center to the circumference, the state of peak separation of each component during development is detected in real time from the center of the disk to the outer circumference, and the corresponding peak separation is performed. By installing a detection unit that emits a signal and a calculation control unit that controls the rotation speed in response to the peak separation signal, we confirmed that each component could be separated well in a short time. The present invention was completed as a result of further research based on these new findings, having confirmed that the method can not only be carried out on an industrial scale but also be automated.

Therefore, the rotary automatic thin layer chromatograph of the present invention has a separation means formed by forming a stationary phase for thin layer chromatography on a disk-shaped substrate, and a rotation driving means for the separation means, a separating section that sequentially develops and separates each component in the sample supplied to the center of the separation means from the center and the center to the outer periphery by rotational driving; a detection unit that detects a peak separation state and generates a peak separation signal corresponding to the peak separation state; and a detection unit that performs a necessary calculation in response to the peak separation signal, and adjusts the rotational speed of the separation means based on the calculation result. The present invention is characterized by comprising an arithmetic control section for controlling.

As the stationary phase, a stationary phase commonly used in chromatography is used, and absorbents, adsorbents, separation agents, etc. are used as appropriate, either alone or in combination with a carrier. For example, the following are used: Nijiri gel, molecular sieves, activated carbon, activated alumina, ion exchange resins, polyvinyl alcohol fibers, synthetic resins, various stationary phase liquids supported on various carriers, and others. As the carrier, commonly used carriers such as diatomaceous earth, crystal, glass beads, and various synthetic trees such as tetrafluoroethylene resin are widely used.

Furthermore, as the stationary phase, in addition to powdered and particulate forms, fibrous forms such as long fibers or short fibers can be used as appropriate. Examples of the fiber material include polyvinyl alcohol, polyethylene, polypropylene, polyacrylonitrile, polystyrene, polyamide, polyester, cellulose, copolymers thereof, crosslinked polymers thereof, and the like. In addition, ion exchange fibers made by introducing cation exchange groups such as sulfonic acid groups and carboxyl groups, or anion exchange groups such as quaternary ammonium groups, for example, sulfonic acid A strong acid type cation exchanger in the form of ultrafine short fibers containing groups can also be advantageously used.

As the structure of the separation means, any structure can be adopted as long as it is of the type that expands from the center to the outer periphery by rotating it. For example, a stationary phase is formed on a disk-shaped substrate. A separation plate type in which the separation agent is accommodated in a space formed between two separation plates via a spacer made of filter paper, filter cloth, etc. can be used as appropriate, and the center part contains the separation agent as a mobile phase. An injection part for injecting the liquid may be provided. The temperature of the stationary phase is maintained at an appropriate temperature by a temperature controller.

In the separation means having such a structure, by rotationally driving the separation means, each component in the sample is separated in concentric circles as the developing solution moves. Alternatively, the stationary phase can be formed in a space with a cross-shaped or rectangular cross section formed using a quarter-circle spacer, or a spiral groove can be cut in the lower disk and the stationary phase can be formed in the corresponding part. You may also As such a means of separation,
For example, commercial products such as rotary centrifugal chromatograph PX-2 (manufactured by Nippon Servo Co., Ltd.) can also be used as is.

In this way, each component in the sample is separated, but in the present invention, it is necessary to detect momentary changes in the separation state in real time. Conventionally known means for detecting the separation state include optical, chemical,
Physical and other detection systems can be used as appropriate, such as infrared spectroscopy, ultraviolet spectroscopy, fluorescence spectroscopy, etc., can also be used advantageously.

In addition, components that are colored on the stationary phase can be detected in their separated state using this device, while non-colored components can be detected by spraying various coloring agents commonly used in thin layer chromatography, or by using various spot detectors. to detect the separation state.

In the arithmetic control section, a known method is used to control the rotational speed of the separating means, such as pulse motor control, sending an instruction signal to a general variable speed motor, or changing the voltage. control is possible.

Since the present invention has the above-mentioned configuration, it can be applied to anything that can be developed in a column, such as organic dyes, various polymer additives, chemicals, biochemical substances, and ionic substances.

Hereinafter, the structure of the rotary automatic thin layer chromatograph of the present invention will be explained in more detail based on the accompanying drawings.

FIG. 1 is a block diagram showing the basic configuration of the rotary automatic thin layer chromatograph of the present invention, in which a separation unit S1 separates each component, a separation unit S1 detects the separation state of each component in the separation means S, and the required It consists of a detection section D that emits a signal, a path for transmitting the signal, and an arithmetic control section C that performs calculations based on the transmitted signal and controls the operation of the separation section.

The operation of each of the above-mentioned parts will be explained in more detail below. The separation section S has a separation means F formed by forming a stationary phase for thin layer chromatography on a disc-shaped base material, and a motor M as a rotational driving means for the separation means, and the separation section S has a temperature The temperature is controlled by a regulator.

The detection section includes a drive section for driving it, a peak detection section, and a position detection section. The peak detection section includes a light source and a light receiving section, and projects light from the light source onto the separating means F, and receives reflected light corresponding to each component on the separating means F at the light receiving section. , and is configured to generate a required peak signal accordingly. Also,
The position detecting section detects the position where the peak detecting section detects the peak, and issues a position signal. Therefore, the detection section detects the peak and position of each component on the separation means F, that is, detects the state of peak separation, and generates a peak separation signal consisting of the peak signal and the position signal. And the calculation control section C
performs a calculation based on the transmitted peak separation signal and controls the rotation of the motor M of the separation section based on the calculation result.

FIG. 2 is a schematic diagram showing the basic structure of the position detecting section, in which the position of the detecting section driven by a motor is detected by a linear sensor and a corresponding position signal is generated.

FIG. 3 shows an example of the peak detection section. This peak detection section consists of a light source, part of a monochromator, and a light receiving section. For example, a deuterium lamp 1 is used as the light source. A part of the monochromator is composed of an entrance slit 2, a rotationally movable diffraction grating 7, a spectrometer collimator mirror 4, and an exit slit 3. The light receiving section is composed of a concave mirror 5, a plane mirror 6, a monitoring photomultiplier 9 equipped with a crystal window plate 8, and a reflection photomultiplier 10, and a driving section ( (see Figure 1).

FIG. 4 shows an example of a movable light receiving section of a spot detection device, in which 11 to 13 are all plane mirrors, and the incident light is read by a monitoring photomultiplier tube 14, while the stationary phase The light reflected from the separated components of the disk F is read by a reflection photomultiplier tube 15, and these two readings are compared to perform quantification and detection. In addition to moving the entire group of mirrors by a drive unit (see FIG. 1), it is of course also possible to move each mirror individually or change their angles.

The peak signal related to the peak waveform detected by the peak detector in this way is as illustrated in FIG.
The signal is greatly amplified by an amplifier circuit, then binarized by a binarization circuit, and then processed by an arithmetic unit. That is, the detected peak signal and the position signal emitted from the position detecting section are subjected to peak separation by, for example, Lorentz curve approximation using an arithmetic element, and are converted into numerical values using a half-value width or an Rf value. As a result, if the separation is good and there are no adjacent components (the Rf values between the spots are far apart), an instruction is given to the rotation control unit to increase the speed, and if the opposite is the case, the speed is decreased, and the separation performance becomes almost stable for each component. Control as follows. In some cases, the components before and after the target component can be discarded without being separated, and in this case, fractionation can be made more efficient.

A known method is used to control the rotation,
For example, control is possible by sending an instruction signal to a pulse motor control, a general variable speed motor, or by changing the voltage.

[For production]

Since the rotary automatic thin layer chromatograph of the present invention has the above-described configuration, each component in the sample is separated by rotating the separation means formed by forming a stationary phase for thin layer chromatography on a disk-shaped base material. is sequentially developed and separated from the center to the outer periphery of the separation means using a developing agent, and the peak separation state of each component is detected in real time by a detection unit, and a corresponding peak separation signal is generated. A necessary calculation is performed in response to this peak separation signal, and the rotational speed of the separating means is controlled based on the calculation result. Specifically, when the detected peak to be separated is far from adjacent peaks (when each component is clearly separated), the separation means is activated by a command from the arithmetic control section. Each component is quickly separated by increasing the rotational speed of the rotating motor. On the other hand, if the peaks to be separated are close to each other, each component is similar and delicate processing is required to separate the two, so such a peak separation signal is input to the calculation control section. If this occurs, the rotational speed of the motor is reduced in response to a command from the arithmetic control section, and each component is completely separated over time.

〔Example〕

Next, dye Solvent consisting of five components of ANE
Using tBlack 3 as a sample and using the device of the present invention,
The separation is shown as an example. Separation by thin layer chromatography and column chromatography is also shown as a comparative example.

Comparative Example 1 Separation of Dye 5 Solvent Black 3 Separation by Column Chromatography ■ Separation by Thin Layer Chromatography Using Merck Art 5789 silica gel 60F-254 as a silicic acid gel preparation plate and toluene as a developing solvent, the dye was separated according to a conventional method. Thin layer chromatography was performed to separate the product into five components having respective Rf values as shown in FIG.

■Separation by column chromatography A sample (dye: S.

1vent Black 3) 300w? Pour the completely dissolved solution into 15ml of a toluene/hexane mixture (1:20), and add silica gel (Merck
Art 7754) and concentrated under reduced pressure using an evaporator to obtain a dried sample-adsorbing silica gel. The column has an inner diameter of 30 mm and is coated with silica gel (Merck Art
7754) To 150 g was added 200 g of a developing solvent toluene/hexane mixture (1:20), air was removed, and the mixture was packed. The above-mentioned sample was placed on top of it and development was started. The developing solvent was a toluene/hexane mixture (1:
20) 4j! It was expanded using.

However, it took approximately 9 hours to separate the components into the five components indicated by ■.

[Example 1] Separation by automatic thin layer chromatography Separation was carried out using the apparatus shown in FIG. 1, which uses a separation means in which a stationary phase for thin layer chromatography is formed on a circular substrate.

First, a sample (Solvent B) is placed in the center on the separation means.
Lack 3) 300 μl completely dissolved in 15 mj of a solvent toluene/hexane mixture (1:20) was placed on the rack and development was started. A toluene/hexane mixture (1:20) 4β was used as the developing solvent, and the separation performance was evaluated using Lorentz curve approximation and Rf values, and the same five components used in the above comparative example were separated. The time could be shortened to about 2 hours.

In other words, when the disk was rotated at 800 rpm for 300 minutes, it was separated into peaks as shown in Figure 6 (a).
First, the first component (component A) was isolated as it was without reducing the rotation speed of 800 rpm.

However, the second . Since the third components (components B and C) were detected to have extremely close peaks, the rotation speed was lowered to 40 rpm to slowly separate them.

After 25 minutes, the two peaks that were close to each other as in (b) could be separated into completely separated peaks as in (b), and the second and third components were successively separated.

As is clear from Figure 6 (b), the 4.5 component is
Although it was not as close as the number 2.3 component, it was detected that it was not possible to completely separate it as it was because they were quite close to each other, so we rotated it for 35 minutes at a rotational speed of 600 rpm+, so that both components were separated as shown in Figure 6 (c). (Component E) was detected to be clearly separated.

In this way, the state of separation of each component, that is, the degree of separation between the peaks of each component, is monitored in real time, and the results are fed back to control rotation, and the state of separation between the peaks of each component is monitored. Appropriate separation was carried out corresponding to the above, and each component was able to be completely separated in an extremely short period of time.

[Effects of the Invention] The present invention includes a detection unit that detects the state of peak separation of each component of a sample on a separation means of a rotary automatic thin layer chromatograph;
By adopting a completely new configuration that organically combines a calculation control unit that controls the rotation of the separation means in accordance with the peak separation state, the speed of separation inherent in rotary automatic thin layer chromatography is improved. This is the first time that we have succeeded in adding the effect of improved separation power to the effect of separation, and it is not only possible to perform complete separation in a very short time, but also to be able to automate it. Because it can be processed, it is possible not only to analyze samples but also to industrially separate and collect target substances.

As described above, according to the present invention, not only the time required for separation is significantly shortened, but also the time required to set conditions is shortened because the separation state can be monitored. As a result, there is no need to repeat trial and error to find conditions, and as a result, sample wastage can be prevented, and even extremely small amounts of samples such as physiologically active substances can be separated without failure, which is extremely useful especially in the technical field of biotechnology. It is.

In addition, since the separation state can be monitored in real time,
For components that are difficult to separate, the rotational speed is lowered, and as a result, it becomes possible to separate components that could not be separated conventionally. In addition, components that are well separated can be separated by simply rotating them at high speed. According to the present invention, not only the separation performance is greatly improved, but also the separation time is shortened. The present invention can also be advantageously used for the separation of biological components that are susceptible to deterioration, and in this respect it will also greatly contribute to biotechnology and the like.

As described in detail above, the present invention is capable of storing biological components, physiologically active substances, dyes specifically exemplified, organic dyes, various polymer additives, drugs, biochemical substances, ionic substances, polymers, etc. in a column. One of the outstanding effects of the present invention is that it can be applied to anything that can be developed, and has a wide range of applications.
It is one.

[Brief explanation of drawings]

FIG. 1 is a block diagram illustrating the basic configuration of the rotary automatic thin layer chromatograph of the present invention, and FIG. 2 is a schematic diagram illustrating the basic structure of the position detection section in the detection section of the chromatograph. FIG. 3 illustrates a spot detection device used as a peak detection section in the chromatograph, and FIG. 4 illustrates another example of the movable light receiving section of the spot detection device. be. Furthermore, FIG. 5 is a thin layer chromatogram of 5olvent Black 3, and FIG. 6 is a diagram illustrating changes in the state of separation of each component of the dye in the rotary automatic thin layer chromatograph of the present invention. . Figure 1 Figure 2 Figure 10 Reflection photomultiplier tube F thin laminate plate Figure 4 Figure 5 (a) During elution of the first component

Claims (1)

[Scope of Claims] 1. Separation means formed by forming a stationary phase for thin layer chromatography on a disk-shaped base material, and rotational driving means for the separation means, and the separation means can be separated by rotationally driving the separation means. a separating section that sequentially develops and separates each component in the sample supplied to the center of the means from the center to the outer periphery; and a separating section that detects the state of peak separation of each component being developed on the separating means, and detects the peak separation state of each component being developed on the separating means. a detection unit that emits a peak separation signal corresponding to the separation state;
A rotary automatic thin layer chromatograph comprising: an arithmetic control section that performs necessary arithmetic operations in response to the peak separation signal and controls the rotational speed of the separation means based on the arithmetic results. 2. The detection section has a peak detection section and a position detection section, and the peak separation signal consists of a peak signal emitted from the peak detection section and a position signal emitted from the position detection section. One term rotary automatic thin layer chromatograph. 3. Control of the rotational speed of the separation means is effective for controlling the rotation speed of the separation means for peak separation signals when the degree of peak separation between two components to be separated and sampled near the outer periphery of the separation means is large. The rotational speed of the separation means is controlled at high speed, and the rotational speed of the separation means is controlled at low speed for a peak separation signal where the degree of peak separation between the two components to be separated and collected is small. A rotary automatic thin layer chromatograph according to any one of claims 1 to 2, characterized in that the rotary automatic thin layer chromatograph is a rotary automatic thin layer chromatograph.