JP2846180B2 - Method for collecting separated components in capillary column liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a capillary column liquid chromatography (Capillary column L).
liquid chromatography, hereinafter simply referred to as CCLC. ))
Specifically, in a CCLC in which a capillary column (separation column, hereinafter, also simply referred to as a column), a detector, and a recorder are combined, a separation component suitable for handling a small amount of an analysis sample (hereinafter, simply, a sample). This is related to the method of sampling.

[0002]

2. Description of the Related Art In general, CCLC is a technique in which a sample is supplied to a capillary column for component separation, and each component in the sample is separated by continuously flowing an eluent (eluate). Usually, the separated components are electrically connected to the column outlet side.
A detector (detector) for detection is arranged , and a recorder is connected to the detector. Each component detected by the detector is electrically measured by a photoelectric tube or the like, and an electrical instruction is transmitted to the recorder. Is continuously recorded as a graph on a recording paper. When the components separated by the CCLC are collected for another test or the like, the eluate passed through the detector is guided to a fraction collector to fractionate the liquid, and a liquid of the separated components is obtained in a test tube of the collector. I have to.

[0003]

SUMMARY OF THE INVENTION However, CCL
When the sample amount handled in C is extremely small, the amount of liquid obtained in one fraction of the fraction collector is, for example, 1 to 50.
It is a difficult problem that it is almost impossible to reliably separate the solution into test tubes. Thus, the present inventor has studied to solve this problem, and as a result, has found a simple method capable of reliably collecting a trace component separated by CCLC, and has achieved the present invention.

That is, an object of the present invention is to separate trace components from a very small amount of sample and to easily separate the separated components.
And reliably collect it is possible to, and easily-obtained was carried out, and in that the handling of the recording paper after treatment provides an easy CCLC separation component collection method was.

[0005]

In order to achieve the above-mentioned object, the present invention provides a capillary column for separating components of a sample, a detector for electrically detecting the components of the separated sample, and an electric power supply for the detector. A recorder for recording the components on the recording paper as a graph by the signal and adsorbing the eluent passed through the detector onto the band-shaped adsorption paper provided on the recording paper, wherein the recorder is provided on the recording paper provided with the adsorption paper. In a CCLC using a process apparatus that moves relative to a sample, a method of collecting a separated component of a sample, in which a sample is supplied to a column filled with a separation carrier and an eluent is continuously flown, and each component in the sample is removed. The separated eluent is used, and the flow rate of the eluent is sequentially sent to the detector as it is, and the separated components are detected by the detector and sequentially recorded on the recording paper of the recorder. While the flow rate, the recording paper strip are sequentially adsorbed by the adsorption sheet arranged to form a linear adsorption portion of the eluent containing the separated components, the linear adsorption unit portion corresponding to the recorded portion of the recording sheet Characterized in that the suction paper containing

The column has an inner diameter of 400 μm to 1.5 μm, for example.
It is formed by filling a separation tube into a small tube of about mm. The separation carrier binds (adsorbs) the components of the sample,
Fine particles having a property of being appropriately eluted by an eluent, for example, silica gel fine particles having a reactive group chemically bonded to a specific substance introduced into the particle surface. The eluent functions to elute the components of the sample adsorbed on the separation carrier of the column. The eluent is, for example, a mixture of two liquids A and B such as water and acetonitrile, and the ratio of the two liquids is adjusted.

The detector is a device for electrically measuring each separated component by light absorption or the like, and uses a device such as a spectrophotometer. The detector sends an electrical signal corresponding to the component measurement to the recorder.

As the recording paper used for the recorder, a movable pen is used to temporarily remove the band-shaped adsorption paper so as not to disturb the space on the paper where the graph is recorded. In addition, the temporary fixing of the suction paper can be performed by appropriately arranging an adhesive tape and an appropriate adhesive at a position avoiding the central portion of the suction paper.

In the detector used in the present invention, the flow rate of the eluent after the electrical measurement is directly adsorbed on the adsorbing paper portion of the recording paper of the recorder. The recording paper is guided and fixed on the suction paper. The recorder has a movable pen that moves relative to the recording paper and records the graph on the recording paper.When connected to the detector, the movable pen is operated properly by the electrical signal of the detector to display the graph on the recording paper. Is recorded continuously. The suction paper is arranged in the traveling direction of the recording paper or the traveling direction of the movable pen.

The adsorbing paper is made of a material which has an appropriate water absorption and does not contain a reactive component, such as a polyvinylidene difluoride (PVDF) sheet, a glass fiber sheet, a nitrocellulose sheet, or a filter paper for chemical analysis. And a sheet cut into a belt shape is used.

In the recording paper, the recording of the eluent component graph and the adsorption of the adsorption paper are performed almost simultaneously. Note that the detector and the recorder are not limited to separate types, and may be of a type integrally formed. Both of them can be used by partially modifying a conventionally known type.

[0012]

The components of the sample are separated in a column.
The total amount of the separated components is sent to the detector according to the eluted flow , detected (measured) by the detector, and continuously recorded on the recording paper of the recorder. On the other hand, the components detected by the detector are guided to the recorder, and are continuously adsorbed on the adsorbing paper attached to the recording paper. The suction paper moves relative to the movable pen (and the discharge port of the small-diameter conduit) together with the recording paper, so that the recording portion of the recording paper and the suction portion of the suction paper have a corresponding relationship. The recording part and the suction part form a set and the recording paper
Provided above. The linear suction portion of the suction paper corresponding to the predetermined recording portion is cut off.

[0013]

Next, an embodiment of the present invention will be described with reference to the drawings. FIG. 1 shows schematic steps of the method of the present example for separating and collecting a separated component of a sample separated by CCLC. In the method of the present embodiment, the column 7 for separating the components of the sample, the detector 10 for detecting the components of the separated sample, and the detected components are recorded as a graph G on the recording paper 23 and the separated components (including the separated components) after the detection of the components are detected. A recording device 20 for adsorbing the eluent) to the adsorbing paper 24 of the recording paper 23 is mainly used, and a process apparatus assembled as shown in FIG. 1 is used. In addition, the liquid tanks 1, 2,
The liquid feed pumps 4A, 4B, the mixer 5, the injector valve 6, the column 7, the detector 10, and the recorder 20 are appropriately connected by capillary tubes 8, 8A (for example, a small-diameter conduit having an inner diameter of 75 μm), and the eluent (A Liquid 3A and liquid B 3B
(Not shown)).

As shown in FIG. 2, the recording paper 23 used in this embodiment is formed by temporarily fixing a strip-shaped suction paper 24 along one side in the longitudinal direction of a normal recording paper. Adsorption paper 24 of this example
In the figure, a PVDF sheet having a width of 5 mm and a thickness of 0.2 mm is temporarily fixed to an appropriate position at the front end in the longitudinal direction and both sides of the width with an adhesive tape 25 so as to be detachable. The temporary fixing portion is provided at a position avoiding the central portion of the suction paper 24. The column 7 comprises a tube having an inner diameter of 320 μm and a length of 15 cm packed with a finely divided separation carrier containing silica gel as a component.

The detector 10 of this embodiment is a spectrophotometer SC-100 (flow cell U, manufactured by Spectra Physics Co., USA).
Z-IL type), the recorder 20 of this example was Unicoder U-228 manufactured by JEOL Ltd., and a part of these was modified and used. That is, as shown in FIG. 1, the detector 10 is connected to the capillary tube 8A after the flow cell 11.
To the recorder 20, and the tube end 22 thereof is connected to the suction paper 24.
Was fixed so as to make light contact with.

An eluent is first supplied to the process apparatus before the sample is supplied. The eluent is composed of a mixture of the solution A and the solution B. During the analysis of the sample, the concentration of the eluate is sequentially increased, for example, at a constant flow rate of 1 to 100 μl / min. In this example, the liquid A 3A in the liquid tank 1 was distilled water, and the liquid B 3B in the liquid tank 2 was acetonitrile. Prior to the supply of the sample, the recording paper 23 is set on the recorder 10, and the detector 10 and the recorder 20 are energized to operate.

Thus, the sample (liquid) (S) containing impurities collected in the syringe 9 is injected into the injector valve 6, and the eluent is kept flowing. The sample arrives at the column 7 together with the eluent, and is adsorbed by the separation carrier to separate each component. The eluate that has passed through the column 7 contains the separated components. Next, as shown in FIG. 1, the flow rate of the eluent
Is the flow cell (measurement cell) of the detector 10 in sequence
11, the light of a predetermined wavelength of the photoelectric tube 12 that has passed through the filter 13 is transmitted through the eluent of the flow cell 11, and the light that has passed through the flow cell 11 is measured for the degree of absorption by the light receiving unit 14, and the recorder 20 , The movable pen 21 is operated, and a graph (chromatogram) G is written on the recording paper 23.

On the other hand, as shown in FIG.
The flow rate of the eluent passed through is sequentially led to the recorder 20 and is adsorbed on the recording paper 24 in the form of a strip. The eluent is solution B 3B
Is sequentially supplied to the column 7 and the separation of the components in the column 7 is continued. The recording of the components on the recording paper 23 and the adsorption of the eluent are continuously performed. Since the recorder 10 of this embodiment is of a type in which the recording paper 23 is sequentially sent forward to the tube end 22 of the capillary tube 8A, the suction paper 2
In 4, the eluent is adsorbed in a continuous linear form,
6 are formed .

Thus, after each component adsorbed on the column 7 is separated, recorded on the recording paper 23 and adsorbed on the adsorption paper 24, the process apparatus is stopped. As shown in FIG. 4, in the recording paper 23 of the recorder 20, a corresponding part 26A of the linear suction part 26 of the suction paper 24 corresponding to the peak GA of the graph G is obtained. That is, the time L1 from the time of injection of the sample to the appearance of the peak GA on the recording paper 23 of FIG.
And the time L2 during which the peak GA occurs is obtained, the positions of the same scales L1 and L2 are obtained from the base point in the linear suction unit 26, and the mark 27 is attached as shown in FIG.
Of the linear suction portion 26 corresponding to the mark 27
By mainly cutting out 6A, the component of the peak GA can be fractionated and collected as a cutout piece 24A as shown in FIG. Similarly, the other corresponding components in the graph G of the recording paper 23 can be obtained by fractionating the components corresponding to the cut pieces from the adsorption paper 24. In addition, the tube end 22 of the capillary tube 8A is connected to the movable pen (pen tip) 21.
In this case, the components in the graph G and the corresponding portion of the linear suction portion 26 of the suction paper 24 can be obtained at a position where there is no time lag. Further, the relationship between the position of the pen tip of the movable pen 21 and the position of the tip end 22 of the capillary tube 8A can be an appropriate correspondence in which the time difference of the flow of the eluent from the flow cell 11 to the adsorption paper 24 can be determined. Then, the separated components of the cut pieces 26A (corresponding portions 26A of the linear adsorption portions 26) are used for other necessary tests such as identification.
The corresponding portion 26A (separated component portion) of the fractionated cut piece 24A is concentrated because the eluent is evaporated,
It is convenient for various tests.

The detector 10 of the above embodiment can be a detector according to the purpose depending on the purpose of the sample and the test. For example, a peptide hydrolyzed by a protease is used as a sample, and after separation by a column, the amino acid primary structure of the peptide can be known by an amino acid sequence analyzer.

[0021]

According to the present invention, the components of the sample separated by the CCLC are detected by a detector and are sequentially recorded on a recording paper of a recorder, and the detected components are sequentially flowed as they are, without changing the flow rate of the recording paper of the recorder. Is continuously absorbed by the suction paper, so that the recording paper is recorded with each separation component, and the separation paper has a linear suction portion in which the separation components are sequentially linearly suctioned in correspondence with the recording. . Therefore, a necessary separation component portion, that is, a separation component, can be easily obtained by cutting out the linear adsorption portion of the adsorption paper corresponding to a predetermined recording portion of the recording paper. And the present invention uses CCLC,
In addition, since the flow rates of the separated components of the sample are successively adsorbed on the adsorbing paper, the entire amount of the trace component can be reliably collected as it is from a small sample or a very small sample. In the present invention, since the components of the sample separated by CCLC are recorded on a recording paper and adsorbed on an adsorbing paper, the present invention can be easily implemented and is convenient. In addition, since the present invention uses a device in which the recorder moves relative to the recording paper provided with the suction paper, the time lag between the graph of the recording paper and the linear suction part is small, and it is easy to correspond the two. .
Further, according to the present invention, since the graph showing the components and the linear adsorption portion of the eluent are formed as a set on the recording paper, unlike the case where the recording paper showing the graph and the adsorption paper are separate, a large number of samples are prepared. When processing is performed, the graph of the recording paper is not mistaken for the adsorption paper on which the eluent is adsorbed, and the processed recording paper is easy to handle.

[Brief description of the drawings]

FIG. 1 is a process diagram of an embodiment of the present invention.

FIG. 2 is a front view of the recording paper before use.

FIG. 3 is a perspective view of a recorder.

FIG. 4 is a perspective view of a recording sheet after measurement.

FIG. 5 is a plan view of suction paper having a linear suction portion.

FIG. 6 is a plan view of the adsorption paper from which a portion having a separation component is cut.

[Explanation of symbols]

 7 Column 10 Detector 20 Recorder 23 Recording paper 24 Adsorption paper 24A Cut-off piece 26 Linear adsorption part 26A Corresponding part G graph

Claims (1)

(57) [Claims] 1. A capillary column for separating components of a sample for analysis, a detector for electrically detecting the components of the separated sample, and a detector for recording the components as a graph on a recording paper by an electrical signal of the detector. And a recorder for adsorbing the eluent that has passed through the belt-shaped adsorption paper provided on the recording paper,
A method for collecting separated components of a sample for analysis in liquid chromatography using a process column in which a recorder moves relatively to recording paper provided with adsorption paper, comprising: a capillary column filled with a separation carrier. The sample for analysis is supplied to the eluent and the eluent is kept flowing.The eluent from each component in the sample for analysis is separated, and the flow rate of this eluent is sent to the detector as it is, and each separated component is detected by the detector. The recording medium is sequentially recorded on the recording paper of the recorder, and the flow rate of the eluent that has passed through the detector is sequentially absorbed as it is on the strip-shaped adsorption paper disposed on the recording paper, and the line of the eluent containing the separated component is sequentially absorbed. the Jo adsorbed portion was formed, separated component collection method capillary column liquid chromatography, characterized in that cutting the absorbent paper comprising a linear adsorption unit portion corresponding to the recorded portion of the recording paper.