JPH02253155A - Preparative chromatography and device thereof - Google Patents

Abstract

PURPOSE:To improve the reliability of refining purity by comparing the absorption spectra of the fraction of a desired component from a sepn. column with the standard pattern of the prescribed desired component. CONSTITUTION:The preparative chromatograph device 1 is set under the same chromatograph conditions as the conditions at the time of a fraction collection and the standard sample of the component desired to be fraction-collected is injected into a sample injecting part 4. The holding time A of the standard sample and the spectra thereof are stored under these conditions. B and C which is % before and after the time A as a basis are set. The measurement time by a photodiode array detector 8 is set at A(1-B) to A(1+C) and the threshold value D for deciding the coincidence with the standard spectra is set. A sample is injected after the setting and the absorption spectra thereof are successively measured. The fraction collection by a fraction collector 7 is started when the coincidence degree of the pattern obtd. in such a manner and the standard pattern is above the value D. The fraction collection is ended at the value D or below. The fraction of the desired component is collected with high purity in such a manner and the reliability of the refining purity is improved.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field This invention relates to preparative chromatography and its apparatus. More specifically, the present invention relates to a technique for fractionating by chromatography in the fields of chemical industry, medicine, food, biochemistry, etc.

(b) Conventional technology In conventional preparative chromatography, fractionation is performed while monitoring the chromatogram, or multiple fractions are collected, and each collected fraction is reseparated to obtain a product with high purity. The fractionated liquids are judged and integrated (called re-chromatography).

Basically, the apparatus for performing the above-mentioned preparative chromatography includes an analysis flow path having a mobile phase supply section, a sample introduction section, a separation column, and a detector in this order, and this flow path is connected downstream of the above-mentioned detector. A fraction collector is used that is capable of fractionating the intended eluate component from the eluate sequentially transferred from the minute needle flow path. The fraction collector of such an apparatus includes a separation nozzle flow path connected to the downstream side of the detector in the analysis flow path via a flow path switching valve such as a three-way valve, and a fraction collector located at the lower stage of the tip of the flow path. It consists of a group of fraction storage bottles prepared in plural numbers.

When performing fractionation using the above device, from the chromatogram detected by the detector of the sample introduced into the analysis channel,
For example, by controlling the flow path switching valve and nozzle flow path in real time in response to chromatographic peaks recognized by monitoring the baseline level and slope, the desired single component can be separated into one bottle. It is configured to be

Furthermore, as an apparatus for performing recycling fractionation, a flow path switching means is attached to the flow path connecting the detector and the fraction collector of the above-mentioned apparatus, and this flow path switching means is connected to the front stage of the separation column in the analysis flow path. It is configured so that the eluate band containing the target component eluted from the column can be reintroduced to the column inlet by connecting the flow channels that communicate with each other. This is equipped with a recycling mechanism to further improve the separation effect.

(c) Problems to be Solved by the Invention However, when conducting an analysis, even if the chromatographs overlap slightly, it is possible to quantify, but when performing a preparative separation, this causes a decrease in the purity of the fraction.

In addition, even if the chromatographic peak on an actual chromatograph is a single component, it is not necessarily close to Gaussian peak due to overload, etc.;
Various peak modifications such as splitting may be performed, or conversely, a peak shape of a single component may be given even though the contaminant components are unseparated. Therefore, it is difficult to accurately judge the degree of purity of the chromatogram from the shape of the chromatogram, and even if the chromatogram is monitored, the purity of both components may be erroneously reduced.

In such a case, subjecting the sample to chromatography again is laborious and time consuming.

The present invention has been made in view of this situation, and it is an object of the present invention to provide a preparative chromatography system and an apparatus therefor capable of reliably collecting both intended components with high purity.

(d) Means for Solving the Problems Thus, according to the present invention, a sample containing a predetermined target component is subjected to liquid chromatography, and the absorption spectrum of at least a fraction of the target component among the sequentially eluted fractions is determined. By monitoring over time and comparing this absorption spectrum pattern with the standard pattern of a predetermined target component at minute intervals, the degree of agreement between the absorption spectrum pattern of the eluate and the above standard pattern can be determined over time. Preparative chromatography is provided, which is characterized in that a high-purity portion in a target fraction is separated by collecting an eluted portion with a degree of coincidence equal to or higher than a predetermined value.

In the preparative chromatography of the present invention, monitoring the absorption spectrum of a fraction of a target component over time means that monitoring is performed in a flow path through which fractions are sequentially transferred. In the present invention, the above-mentioned monitoring is performed by detecting the spectral pattern of the fraction intended for fractionation, and a photodiode array detector is suitable for this purpose. The above-mentioned monitoring detector is provided at the downstream stage of a liquid chromatography separation column in a flow path through which fractions sequentially eluted from the column are transferred.

The above monitoring may be performed continuously until the liquid chromatography is completed on the injected sample, or at least on a fraction of the target component. In the latter case, the monitoring time is usually set within a predetermined range before and after the retention time of the sample of the target component.

The monitored spectral pattern is compared with a standard pattern at every minute time. It is preferable that the above-mentioned minute time is a time interval as short as possible, but it is subject to constraints on the equipment that implements this method as described later, such as the processing speed of calculations, for example, 0. O1-1 sec is preferable.

In the above comparison, 1. The degree of matching of the patterns is determined.

The degree of matching can be grasped by directly expressing the degree of correspondence between two waveform patterns based on a technique such as waveform analysis. Therefore, it is possible to set a predetermined value (a so-called threshold value) for this obtained numerical value, and to judge that a match is equal to or greater than this predetermined value = 7. For details, refer to the description of the embodiment described later. By collecting a fraction with a predetermined value or more, the target component can be isolated with high purity.

Therefore, the present invention also provides an elution channel having a mobile phase supply section, a sample introduction section, and a separation column in this order, as a suitable apparatus for carrying out the above-mentioned preparative chromatography, and an elution channel attached to the channel and sequentially transferred through the channel. a fraction collector section configured to collect a fraction containing a predetermined target component from the eluate to be extracted; (b) a photodiode array detector capable of monitoring over time the absorption spectrum of at least a fraction of the target component of the eluate sequentially transferred through the flow channel; and (b) communicating with the elution flow channel after the detector. (c (d) a comparison unit that compares the spectral pattern monitored by the photodiode array detector with the standard pattern stored in the storage unit at minute intervals; , (e) a determining unit that determines the degree of pattern matching based on signal values sequentially output from the comparing unit;
f) A preparative liquid chromatography device comprising a fraction collector operation control unit that instructs a fraction collector to operate so as to collect the eluate portion for which the degree of coincidence is determined to be equal to or higher than a predetermined value by the determination unit. can be provided.

In the above apparatus, the elution channel having a mobile phase supply section, a sample introduction section, and a separation column in this order can be one known in the art as it is.

The fraction collector section attached to the above elution channel is
Specifically, a fraction collector, a communication channel that communicates this with the elution channel, a photodiode array detector provided in the communication channel, and an output signal from the detector is processed to operate the fraction collector. operating memory,
It is composed of a comparison section, a judgment section, and a fraction collector operation control section.

The communication flow path prevents deviations in fractionation due to a time lag in which a decision is made by the decision section based on the detection signal output from the detector and an operation signal is output from the fraction collector operation control section based on this decision. Therefore, it is preferable to suppress the diffusion of the eluate band transferred from the elution channel and have a constant volume. For example, a hollow conduit having a constant diameter and a constant length, a column-shaped conduit filled with microparticles, etc. are preferable. used.

As the fraction collector, any structure known in the art can be used, except that it is configured to allow fractional supply based on a control signal from a fraction collector operation control section.

It is preferable that the storage section, the comparison section, the judgment section, and the fraction collector operation control section are configured as a signal processing section using a CPU. In the signal processing section, fractionation is started for the eluate whose degree of coincidence is determined to be greater than a predetermined value as described above through a series of signal processing by the storage section, comparison section, and judgment section, and when this degree of coincidence is greater than the predetermined value. The fraction collector is configured to be able to output a control signal instructing the fraction collector to operate so as to terminate the fractionation when the fraction collector becomes smaller.

Further, according to the present invention, a flow path switching means is further provided in the flow path between the photodiode array detector and the fraction collector in the fraction collector section of the device,
A return channel is provided that communicates with at least the upstream stage of the separation column in the elution channel from the channel switching means, and a portion of the eluate whose degree of coincidence is determined to be smaller than a predetermined value by the determining section can be transferred to the return channel. There is provided a preparative liquid chromatograph apparatus further comprising a flow path switching operation control section capable of switching and controlling the flow path switching means.

In the above apparatus, the flow path switching means is provided in the communication flow path between the elution flow path and the fraction collector section of the preparative liquid chromatography apparatus described above. Further, one end of the return flow path is connected to the flow path switching means, and the other end of the return flow path is connected to at least the upstream stage of the separation column in the elution flow path. In this case, it is preferable to connect between the mobile phase storage tank and the liquid feeding means in a normal mobile phase supply section. The basic structure of the flow path configuration provided with the above-mentioned flow path switching means and return flow path is that of an apparatus configured to enable recycling fractionation known in the field, especially an apparatus for peak shaving recycling fractionation. It can be used as

The device of the present invention includes the above-described fraction collector section in the basic flow path configuration, and is configured such that switching of the flow path switching means is operated based on the determination section of the fraction collector section. This switching is performed at a timing when the eluate portion whose degree of coincidence is determined to be smaller than a predetermined value by the determination section can be transferred to the return channel.

(Left below) (E) Effect: According to the present invention, the absorption spectrum of at least the fraction of the target component among the fractions sequentially eluted from the separation column is monitored over time, and the pattern of this absorption spectrum at every minute time is monitored. is compared with a standard pattern of a predetermined target component, the degree of matching is determined over time, and only the eluted portions with this degree of matching equal to or higher than a predetermined value are collected.

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereby.

(F) Examples Example 1 FIG. 1 is an explanatory diagram of the configuration of an example of a preparative liquid chromatograph apparatus of the present invention. In the above figure, a preparative liquid chromatography device (1) has an elution channel (a ), and a fraction collector section (6) attached to the flow path (a) and configured to collect a fraction containing a predetermined target component from the eluate sequentially transferred through the flow path. There is.

The separation column (5) is a preparative column with a packing material (ODS particle size of 15 μm), a diameter of 50 mm, and a column length of 25 cm.

The fraction collector section (6) includes a fraction collector (7), a communication path (b) that communicates this with the elution channel (a), and a photodiode array detector (8) provided in the communication path (b). ) and a signal processing section (9).

The communication path (b) is configured to suppress the diffusion of the eluate bands that are sequentially separated and transferred in the elution channel (a) and to have a constant volume. For example, as shown in Figure 2, there are hollow pipes with a constant diameter and length (see figure (a)), and column-shaped tubes filled with microparticles such as stainless steel spheres and glass beads (particle diameter of 10 to 100 μm). Pipeline (same figure (b))
etc. The above-mentioned volume is designed to correspond to the product of the time lag from the output of the detector described below to the operation of the fraction collector and the flow rate of the eluate transferred through the communication path.

The fraction collector (7) includes a flow-out channel connected to the communication path (b), a group of preparative ports optionally connected to the flow channel, and fraction storage ports prepared corresponding to these port groups. It consists of a group of bottles.

The photodiode array detector (8) has a light source that irradiates light over the entire wavelength range and about 500 photodiodes, and detects at least the target component of the eluate band transferred through the communication path (b). The system is configured to be able to monitor the absorption spectra of the bands over time.

The signal processing section (9) includes a built-in CP, a U (91), a storage section (92), a comparison section (93), a judgment section (94), and a fraction collector operation control section (95). It is arranged to process the output signal from the photodiode array detector (8) to operate said fraction collector (7). That is, the storage unit (92) stores in advance the spectral pattern of the standard sample of the target component (hereinafter referred to as standard pattern), and the comparison unit (93) stores the spectral pattern output from the photodiode array detector (8) in advance. The signal is compared with the standard pattern stored in the storage unit at approximately every 0.1 sec, and the determining unit (94) determines the pattern based on the signal values sequentially output from the comparing unit at approximately 0.1 sec intervals. The fraction collector operation control section (95) controls a predetermined port of the fraction collector (7) so as to collect the eluate portion whose degree of coincidence is determined to be equal to or higher than a predetermined value by the determination section. The device is configured to command the operation of connecting the .

The judgment unit (94) calculates the degree of coincidence of the spectral patterns between the two absorption spectra S+(λ), S2(λ) (λ wavelength) based on the following formula,
The threshold value (
D), and when the calculation result is equal to or higher than this threshold value (D), a signal is output to the fraction collector operation control section (95). Note that the threshold value (D) used is approximately 0.99 to 0.9999.

The preparative liquid chromatography device configured as above (
The operation of 1) will be explained.

l) First, set the same chromatographic conditions as during preparative collection, and inject a sample of the component of the preparative port into the sample injection port. The retention time (A) of the standard specimen under these conditions and its spectrum (standard spectrum, S) are memorized.

2) Based on the above retention time (A), calculate the percentage before and after (B)
), (C) as appropriate to adjust the measurement time by the photodiode array detector (8), for example, from A(1-B) to A.
(1+c), and on the other hand, a threshold value (D) for determining the degree of matching with the standard vector.

After the above settings, inject the sample and 0.1 within the above measurement time.
Absorption spectra are measured sequentially at intervals of sec, and the degree of agreement with the standard pattern is calculated for the sequentially obtained spectral patterns, and this degree of agreement is set as a threshold value (D
) With the above steps, start fractionation by fraction collection, and (
D) End the fractionation with: A flowchart of an example of the above operation is shown in FIG.

In addition, in the above device (1), the set time A (IB) ~
If a fraction equal to or higher than the threshold value (D) cannot be obtained within A (1-C), or if the fraction is too small, the fraction equal to or lower than the threshold value (CD) is divided into another fraction within the set time. If the configuration is such that it can be retained in the image port, it will be convenient to be able to roughly separate it when it is re-injected and re-separated.

As described above, according to the above-mentioned apparatus (1), it is possible to separate a fraction of the target component with high purity.

Embodiment 2 FIG. 4 is an explanatory diagram of the configuration of another example of the preparative liquid chromatography apparatus of the present invention. The device (10) in this figure is the same as the device (1) in Example 1 above, but has a three-way connection in the communication path (b) between the photodiode array detector (8) in the fraction collector section (6) and the fraction collector. solenoid valve(
11) is provided, and an elution channel (
It is constructed by providing a recycle channel (c) between the mobile phase storage tank (2) of a) and the liquid sending pump (3). The three-way solenoid valve (11) has two sides (hereinafter referred to as flow-out position) that communicate the communication passage (b) with the fraction collector and a side (hereinafter referred to as flow-out position) that communicates the communication passage (b) with the recycling passage (c).
It is configured so that it can be switched and connected to the recycle position (hereinafter referred to as the recycle position).

The signal processing unit (9) is further provided with a three-way solenoid valve control unit (96), and the eluate portion whose degree of coincidence is determined to be smaller than a predetermined value by the determining unit (94) is transferred to the recycling channel (
The three-way solenoid valve control section (96) is configured to output a signal for switching and controlling the three-way solenoid valve (1) so as to transfer to the three-way solenoid valve (96).

Other than the above configuration, the configuration and functions of the first embodiment are maintained as they are.

Next, the operation of the preparative liquid chromatography apparatus (10) configured as described above is performed for the eluted components 1 and 2 whose chromatographic peaks overlap. The case where peak shaving recycling fractionation is performed for n (where the earlier elution is assumed to be ■) will be explained as an example.

l) First, set the same chromatographic conditions as during preparative collection, and inject the standard sample (■) of the component (2) to be separated into the sample injection section. At this time, the retention time (A) and its spectrum (standard spectrum, SA) of the standard specimen (■) are memorized.

2) Under the same conditions, press-fit the sample (2) of the component (2) to be separated, and memorize the retention time (B) and its spectrum (standard spectrum, SR).

3) The above retention times (A) and (B) and the recycling flow path (
c) is transferred from the time h (h: the recycling channel volume divided by the flow rate), the retention time for the Xth recycling is t n1x = A K + h (x 1 ) tR11, respectively.
x=Bx+h(x-1).

The operating conditions of the photodiode array detector (8) are set based on the above holding time.

■Measurement time starts 0 hours earlier than retention time tR1x,
Repeat ending 2 hours later than t+lx.

(2) Set a threshold value F1 for determining the degree of agreement with the standard spectrum (SA) and a threshold value G for determining the degree of agreement with the standard spectrum (S8).

■Ingredients of fraction collector (7)■Poto (H),
Set the port (r) for component (2).

■Set the peak shaving recycle fractionation end time J.

After setting the above conditions, the three-way solenoid valve (11) is set to the flow-out position, the sample is injected, and the absorption spectra are measured sequentially every 0.1 sec within the measurement time predicted from the retention time of each target component. Take measurements.

The degree of agreement with the standard pattern (SA) for the sequentially obtained spectral patterns is calculated using the formula in Example 1, and when this degree of agreement is greater than or equal to the threshold value (F), fractionation is performed by the fraction collector port (H). Begins.

The degree of matching obtained by the above calculation is the threshold value (F')
, this signal is output to the three-way solenoid valve control (96), which switches the three-way solenoid valve (11) to the recycle position.

After the above-mentioned time point, the degree of coincidence of the sequentially obtained spectral patterns with respect to the standard pattern (SB) is calculated in the same way.

When the degree of coincidence obtained by the above calculation exceeds the threshold value (G), this signal is output to the control section (96), and the control section (96) switches the three-way solenoid valve (11) to the floor position and closes the fraction collector photo. Fractionation is started by (■), and the above match is the threshold value (G).
The fractionation is terminated when the value falls below.

The above series of operations is performed for a holding time t within the set time J.
RIX, t Due to the relationship with RIX, it will be repeated for the eluate to be cycled X times. FIG. 5 shows a schematic diagram of the chromatograms of components I and H for the Xth time of recycling.

Incidentally, an example flowchart specifically showing the above operation is shown in FIG. 6.

From the above, according to the device (10), when performing peak shaving recycling to separate and fractionate target components (2) and (3) whose elution positions overlap in a short time, the fractionation timing and recycling timing are automatically determined. By doing so, highly purified fractions can be obtained.

(G) Effects of the Invention According to the present invention, since the purity level of peaks that cannot be judged from the chromatogram can be determined and fractionated, the reliability of the purification purity can be improved. Furthermore, target components whose elution positions overlap can be purified and fractionated with high purity. Furthermore, it is possible to eliminate the need for setting the fraction collector itself.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the configuration of an example of a preparative liquid chromatograph apparatus of the present invention, FIG. 2 is an explanatory diagram of an example of the configuration of a communication path in the apparatus of FIG. 1, and FIG. FIG. 4 is a flowchart diagram showing an example of the preparative liquid chromatography apparatus of the present invention; FIG.
The figure is a schematic diagram showing a chromatogram of components (1) and (H) for the Xth time of recycling, and FIG. 6 is a flowchart diagram showing an example of the preparative separation operation of the apparatus of FIG. 4. (2)...Mobile phase storage tank, (3)...
Liquid sending pump, (4)...Sample injection part, (5)
... Separation column, (6) ... Fraction collector section, (7) ... Fraction collector, (8) ... Photodiode array detector, (9) ...
...Signal processing section, (11) Old three-way solenoid valve, (
91)...CPU. (92)...Memory Department, (93)...Dan...
Comparison section, (94)... Judgment section, (95) Fraction collector operation control section, (9
6)... Three-way solenoid valve control section, (a)... Elution channel, (b) Old... Communication channel, (e)... Recycling channel. ■ Procedural amendment run E+ (method) Display of the case 1999 Patent Application No. 75650 2, Name of the invention Preparative chromatography and its apparatus Representative Nishijojo Minoru Date of amendment order July 4, 1999 (all sent) (Japanese) Figure 6 of the drawing subject to amendment

Claims (1)

[Claims] 1. A sample containing a predetermined target component is subjected to liquid chromatography, and the absorption spectrum of at least a fraction of the target component among the sequentially eluted fractions is monitored over time, and By comparing this absorption spectrum pattern with the standard pattern of a predetermined target component each time, the degree of agreement between the absorption spectrum pattern of the eluate and the above-mentioned standard pattern is judged over time, and if this degree of agreement is greater than a predetermined value. Preparative chromatography is characterized in that a high purity fraction in a target fraction is separated by collecting an eluted fraction. 2. An elution channel having a mobile phase supply section, a sample introduction section, and a separation column in this order, and collecting a fraction containing a predetermined target component from the eluate attached to the channel and sequentially transferred by the channel. (a) The fraction collector section is installed after the separation column in the elution channel, and is configured to collect at least the target component of the eluate sequentially transferred through the channel. (b) a photodiode array detector capable of monitoring absorption spectra of fractions over time; a fraction collector consisting of a port group that can be supplied separately and a plurality of fraction storage bottles prepared below the port group; (c) a storage unit that stores in advance a standard pattern of the target component; (d) ) a comparison section that compares the spectral pattern monitored by the photodiode array detector with the standard pattern stored in the storage section every minute time; (e) based on signal values sequentially output from the comparison section; and (f) a fraction collector operation control unit that instructs the fraction collector to operate so as to collect the eluate portion for which the degree of matching is determined to be equal to or higher than a predetermined value by the determination unit. A preparative liquid chromatography device comprising: 3. A flow path switching means is provided in the flow path between the photodiode array detector and the fraction collector in the fraction collector section of the apparatus according to claim 2, and from this flow path switching means to at least the upstream stage of the separation column in the elution flow path. a flow path switching operation control capable of controlling flow path switching means to provide a return flow path communicating with the flow path, and to transfer a portion of the eluate whose degree of coincidence is determined to be smaller than a predetermined value by the determination unit to the return flow path; A preparative liquid chromatography device further comprising: