JPS61140861A - Liquid chromatography - Google Patents

Abstract

PURPOSE:To analyze surely an intricate material or a mixture composed of materials having resembling structures by mixing such a solvent which concentrates components with the eluate from a column and introducing the same into a concentrating column. CONSTITUTION:A valve 1 is set to flow passage shown by solid lines. THF (tetrahydrofuran) in a vessel 2 is passed to a sample introducing pipe 5 and the sample is introduced therein. The THF dissolved therein with the sample is introduced into a GPC column 6 and after the sepn. of the sample, the eluate is run to a detector 7 and pipes 10, 11. The prescribed GPC eluted fraction is stored in a storage loop 9. The valve 1 is then set to the flow passages shown by dotted lines and valves 12, 15 are set to the flow passages shown by solid lines to pass the THF to pipes 5, 10, 13, 16. The diluting water in a vessel 17 is run to pipes 19, 16 and is mixed with the THF and thereafter the mixture is run to pipes 21, 28. The GPC eluted frac tion is concentrated in an ODS column 20 for concentration. The valve 12 is thereafter set to the flow passages shown by the dotted lines to pass only the diluting water, thereby replacing the THF in the column 20. The valve 15 is then set to the flow passages shown by the dotted lines to pass the eluate in a vessel 22 and to elute the concd. sample in the column 20. The sample is introduced into a column 25 and is subjected to analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to liquid chromatography, and more specifically, to first columns having different separation modes, such as an organic solvent-based GPC column and a reversed phase distribution type column. and a method for separating components by connecting a second column.

Conventional techniques and their problems When analyzing a complex mixture of substances or a mixture of substances with similar structures by liquid chromatography, it may not be possible to completely separate each component in the sample using a single separation column. In this case, the first and second columns, which have different separation modes, are connected to separate the components. For example, water-based GP is a conventional method for analyzing biological samples.
A method using liquid chromatography in which a C column and a reverse phase distribution column such as an ODS column are connected has been proposed, and a biological sample having a complex composition is separated into its components by this method.

In the above method, the eluent of the aqueous GPC column?
It is directly introduced into a reversed phase distribution type column, and in this case relatively good peaks can be obtained. However, when trying to apply this method to the analysis of industrial chemicals, many industrial chemicals have low solubility in water;
It is necessary to use an organic solvent-based GPC column, but when the eluate from an organic solvent-based GPC column is directly introduced into a reversed-phase partition column, good peaks cannot be obtained as shown in their experimental example. There is a problem.

The present invention was developed in view of the above circumstances, and is based on the above-mentioned FjN organic solvent-based GPC column. In liquid chromatography, in which components are separated by connecting a first column and a second column with different separation modes, such as a reversed-phase partition type column, it is possible to obtain good separation of each component, and therefore, it is possible to obtain a good separation of each component. In order to ensure complete analysis of mixtures or mixtures of substances with similar structures, a concentration column is interposed between the first column and the second column, and this eluate is added to the eluate from the first column. Mix all the solvents to be concentrated in the above concentration column, introduce this mixture into the above concentration column, concentrate the above components in the concentration column, and then transfer the concentrated components in the concentration column to this @condensation column. The concentrated component in the concentration column is introduced into the second column through the solution that is eluted and acts as an eluent for the second column.

Next, the present invention will be specifically explained by showing examples.

Example FIG. 1 is an example of a multidimensional mode high performance liquid chromatograph used to carry out the liquid chromatography of the present invention. It shows. In the figure, 1 is a four-way valve (first valve) having eight passage ports 1a to 1h2, and the passage port 1 of the hexagonal pulp 1.
One end is connected to a THF (tetrahydrofuran) tank 2, and the other end of a sample introduction tube 5, in which a THF liquid feed pump 3 and a sample inlet 4 are interposed, communicates with a. In addition, the passage port 1bK communicates with one end of a first separation channel pipe 8 in which an organic solvent-based GPC column (first column) 6 and a UV detector 7 for monitoring GPC eluate are interposed.
The other end of the flow path pipe 8 communicates with the passage port IC. One end of a sample storage channel tube 10 in which a capacitance 2-〇 PC elution fraction storage loop 9 is interposed is communicated with the passageway opening 1d, and the other end of the flow channel tube 10 is connected to the channel opening 1d. Road entrance 1h
is connected to. Note that the passage port 1f is closed, and the other end of the first discharge pipe 11, one end of which is open to the outside of the system, communicates with the passage port 1g. A four-way valve (second pulp) having a passage opening 12a of the four-way knob 12 and a passage opening 1e' (!: of the six-way knob 1) are not connected by the first communication channel 13. Note that the passage port 12b communicates with the other end of the second discharge pipe 14, one end of which is open to the outside of the system, and the passage port 12C is closed.

Furthermore, 15 is a six-way valve (third valve) having six passage ports 15a to 15fi, and a second communicating flow pipe is connected between the passage port 15a of the six-way valve 15 and the passage port 12d of the four-way valve 12. 16. In addition, one end of this second communication channel pipe 16 is connected to a dilution water tank 17 for concentration, and the other end of a dilution water introduction pipe 19 in which a dilution water supply pump 18 for concentration is interposed is connected. Furthermore, one end of a sample concentration flow pipe 21 in which an ODS column 20 for GPC elution fraction concentration is interposed is connected to the passage port 15c, and the other end of this flow pipe 21 is connected to the passage port 15f. It's communicating. One end of the passageway port 15d is connected to the ODS eluent eluent (cetonitrile-water, etc.) tank 22.
The other end of the 7j ODS eluent inlet pipe 24 is connected to the S eluent eluent feeding pump 23. One end is open to the outside of the system and the other end of a second separation channel pipe 27 is connected to the passage port 15e, and the other end is connected to a reverse phase distribution type OD8 column 25 and a UV detector 26 for monitoring ODS eluate. .

Note that the other end of a third exhaust pipe 28, one end of which is open to the outside of the system, communicates with the passage port 15b.

29 and 30 are recorders connected to the detectors 7 and 26, respectively, and 31 is a control device connected to the pumps 3 and 18 to control the flow rates of these pumps 3 and 18.

When analyzing a sample using the above-mentioned apparatus, first set the flow path state of the four-way valve 1 as shown by the solid line in the purple diagram at the flow path opening 1a.
and lb, lc and ld, le and if.

1g and lhi communicate. Then, in this state, TH
The F liquid feeding station 3 is fully activated to flow all THF into the THF sample introduction tube 5, and at the same time, the sample is introduced from the sample injector 4 into the sample introduction tube 5. Then, the passage port 1a is inserted into THF' in which the sample is dissolved.

1bv! - through the first separation tube 8, introduced into the GPC column 6, where the sample is separated;
Flow through the flow path of the passage port 1h, Ig, and the first discharge pipe 11,
During this time, a predetermined GPC elution fraction is stored in the storage loop 9.
to be stored.

Next, the flow path states of the four-way valve 1 are shown by dotted lines in the figure: passage ports 1b and lc, ld and 1e, and lf and Ig.

1h and lai, and the four-way valve 12 is shown as a flow path in the figure with passage ports 12b, 12C, 912d, and 12a.
The passages 1b and lc, ld and le, and lf and 1aQ are in communication with each other, with the six-way valve 15 in a flow path state shown by the solid line in the figure. In this state, fully operate Denji 3 and TI (F'
e After passing through the sample introduction tube 5, passage ports 1a, 1h2, and flowing into the channel tube 10, the sample is further passed through passage ports 1d, 1e,
The water passes through the first communication channel pipe 13, the passage ports 12a and 12di, and flows into the second communication channel pipe 16. Then, the pump 18 is operated to flow all of the dilution water in the dilution water tank 17 from the dilution water inlet pipe 19 to the second communication channel pipe 16, and after completely mixing the dilution water with THF, this mixed liquid is poured into the passage ports 15a and 15f. , the flow path pipe 21 for sample concentration, the passage ports 15c and 15b, and the third discharge pipe 28, thereby directing the GPC elution fraction to the OD for concentration.
Concentrate into S column 20.

After that, the four-way valve 12 is switched, and the passage ports 12a and 12b are set to the flow path state shown by the dotted line in the figure.

12c and 12di communicate. Then, only the dilution water was passed through the flow path mentioned above, and the THF'
? Replace with dilution water.

Next, the channel states of the 15 six-way valves are shown by dotted lines in the figure, with passage ports 1a and lh, lc and ld, and le.

Connect 1f. In this state, the Imp 23
The eluent in the ODS eluent tank 22 is transferred from the eluent inlet pipe to the passage ports 15d and 15c.

The concentrated sample in the concentration column 20 is eluted by the eluent, which is passed through a flow path including the sample concentration flow path tube 21, the passage ports 15f and 15e, and the second separation flow path tube 27, and this is subjected to reverse phase distribution. The sample is introduced into a type ODS column 25 and analyzed.

In the embodiments described above, W solvent (THF)-based GP
The C column 6 and the reversed phase distribution type ODS column 25 are connected to O for concentration.
In addition to connecting the DS column 20vf as an interface, the eluate from the GPC column 6 is mixed with dilution water, and this mixture is introduced into the column 20 for total concentration.
After concentrating the eluted fraction of the column 6 in the concentration column 20, it is replaced with THFi water in the concentration column 20, and then the ODS eluate is passed through the concentration column 20, and the concentrated components in the concentration column 20 are Since it is introduced into the eODS column 25, industrial chemicals containing a mixture of complex components can be well analyzed.

Next, experimental examples will be shown to specifically explain the effects of the present invention.

Experimental Example 1 The following samples ① to ② were separated using only a reversed phase partition type ODS column. The results are shown in Figure 2 - 0.

Sample ■: 11000 pp of benzene and 500 pI)
THF solution containing 10 ppm of toluene (Figure 2 (4)) Sample ■: 10 ppm of 2, . THF solution containing 4-6-drimethylphenol and 10 ppm p-5ee-butylphenol (Figure 2 (3)) Sample ■: 1000 m) l) m benzene, 500 p
A THF solution containing pm of toluene, 10 ppm of 2.4.6-)limethylphenol, and 10 ppm of p-5ec-butylphenol (Figure 2 0,) Results shown in Figure 2 - 0! In the J, ODS column, it was observed that penzene, 2.4.6-)limethylphenol, and doruendo p-5ec-butylphenol were separated.

Experimental Example 2 The above sample (2) was separated only using an organic solvent (T, F (F)) based GPC column. The results are shown in FIG.

From the results shown in Figure 3, it was confirmed that benzene and toluene could not be separated using the GPC column.

Experimental Example 3 Using the apparatus shown in FIG. 1, the above-mentioned sample (1) was completely separated by the method described above. The results are shown in Figure 4. The analysis was performed according to the following procedure.

(1) Introduce a 20 μt sample into the papc column 6.

+2112 minutes later, THF liquid pump 3 k 0.4++
+4'd, dilution water pump 1 B'< 3.6 m
The liquid is sent to the concentration column 20 for 3.5 minutes at a flow rate of 3.5 minutes.

(3) Switch the 12 square pulps and send only water to the concentration column 20 for 40.5 minutes.

(4) Replace the six-way valve 158 and introduce the entire sample concentrated in the concentration column 20 into the ODS column 25. At the same time as the sample is removed, the recorder 30 connected to the UV detector 26 for monitoring the ODS eluate is started.

According to the method of the present invention, the results shown in Fig. 4 are as follows: 2,4.6
It was proposed that it would be possible to separate benzene and toluene in a sample containing two phenolic substances, -dolimethylphenol and hypo-5ec-butylphenol, as impurities.

Experimental Example 4 Using the apparatus shown in FIG. 5, the ODS elution pattern was investigated when the benzene and toluene eluted portions of S-3 in FIG. 3 were directly introduced into an ODS column without being concentrated in a concentrating column. In the apparatus shown in FIG. 5, parts having the same configuration as those in FIG. Moreover, the analysis was performed according to the following procedure. The results are shown in Figure 6.

(1) Input valve 1? Il - the flow path state shown by the solid line,
By operating the pump 3, the above sample (2) of 20 μt is introduced from the sample injector 4 into the GPC column 6.

After +2112 minutes, the manual valve 1 is fully switched to the flow path state shown by the dotted line, and the pump 23 is activated to introduce the GPC elution fraction 'kODs column 25 in the storage loop 9 and ODS.

Recorder 30 connected to UV detector 26 for monitoring eluate
Gold starts.

+31 1.2 minutes later, input pulp 1? Switch to return to the flow path state shown by the solid line.

From the results in Figure 6, the elution fraction of the GPC column?
It was observed that benzene and toluene were not separated when they were introduced directly into an ODS column without being concentrated in a concentrator column.

Experimental Example 5 Using the apparatus shown in FIG. 1, the influence of the mixing ratio of TI(F2: dilution water) upon total concentration of the sample in the concentration column 2o was investigated.

In this case, the GPC elution fraction (2 ml as Denji 3 flow rate) was appropriately mixed with water (by adjusting the flow rate of the pump 18) and introduced into the concentration column 20.

This was eluted with ff1ODs eluent and separated using ODS column. The relationship between the obtained peak height and the THF concentration during concentration is shown in FIG.

From the results shown in FIG. 7, it was found that in all samples, the abrasive peak was higher when the THF concentration was lower.

Further, the peak shape of m-xylene at this time is shown in FIG. 8, and as a result, the higher the THF concentration, the sharper the distortion.

This increase in peak distortion and the accompanying decrease in peak height is due to the fact that THF in the concentration column greatly disturbs the eluent composition at the moment the sample is charged at the tip of the ODS column.
It is presumed that this is caused by changing the distribution of the sample.

Experimental Example 6 Based on the estimation in Experimental Example 5, the influence of replacing the inside of the concentration column with water after concentrating the eluted fraction in the concentration column on the peak height and shape was investigated.

As a result, as shown in FIG. 9, it was found that at any THF concentration, a substantially constant peak height was exhibited at displacement water fl m1 or higher. However, as shown in Figure 10, if 1 to 2 ml or more of replacement water was used, the distortion of the peaks was almost eliminated.

Experimental Example 7 From the results of Experimental Example 6, it was found that the highest peak height could be obtained by setting the water exchange amount f1ml or more. We investigated the effect of sound on the collection efficiency of T H,F concentration during concentration in a concentration column (6wnφxaomm).In this case, toluene and The changes in peak height and collection efficiency of m-xylene and mesitylene were all measured.The results are shown in Figures 11 and 12.

From the results in FIG. 11, it can be seen that the lower the THF fa degree is, the higher the peak attack is, and that even at 10% THF/water, there is still an upward trend. Also, from the results in Figure 12,
The collection efficiency varies slightly due to the difference in hydrophobicity of each sample, but all samples have almost constant values up to 40% THF/water. It is recognized that it is shown. From this, 40% THF/
It has been found that if the THF concentration is lower than water, almost the entire amount is collected in the concentration column.

Experimental Example 8 Next, what is the size of the concentration column? Effects on peak height and collection efficiency when changed were investigated in the same manner as in Experimental Example 7. In this case, the concentration column should be one with 6 holes x 30 holes or one with 6 holes x 50 holes? Using. result? 13th
It is shown in FIG.

As a result, as shown in Fig. 14, by setting the length of the concentrating column to 50 cm, a collection efficiency of approximately 100 cm can be obtained with a 60% THF/water trench, but as shown in Fig. 13. It was observed that the peak height was not much different from that of the 3° flare.

Effects of the Invention As explained above, the liquid chromatography according to the present invention has a first column and a second column that have different separation modes.
In liquid chromatography in which components are separated by connecting columns, a concentration column is interposed between the first column and the second column, and the components in this eluate are added to the eluate from the first column. Mix the solvents that will be concentrated in the concentration column, and introduce the entire mixture into the concentration column to concentrate the above components. Concentrated components in the concentration column through a solution that both elutes and acts as an eluent for the second column? By introducing it into the second column, p
Even when analyzing a complex mixture of substances or a mixture of substances with similar structures, each component can be separated and good peaks can be obtained for each.

[Brief explanation of the drawing]

Figure 1 is an example of a multidimensional high-performance liquid chromatograph used to carry out the liquid chromatography of the present invention. The schematic diagram shown in Figure 2 - O is a chromatogram of a sample analyzed using a reversed-phase partition ODS column, and Figure 3 is an organic solvent-based GPC.
Fig. 4 is a chromatogram obtained by performing four analyzes of a sample using the column, Figure 4 is a chromatogram obtained by performing four analyzes of a υ sample using the apparatus shown in Fig. 1, and Fig. 5 is a liquid chromatogram used in Experimental Example 4. Figure 6 is a chromatogram obtained by analyzing a sample using the apparatus shown in Figure 5, and Figure 7 shows the THF concentration and peak in dilution water when analysis is performed using the apparatus shown in Figure 1. A graph showing the relationship with height, Figure 8 shows the TI for dilution water using the apparatus shown in Figure 1.
(F concentration? Chromatogram of all samples analyzed while changing the concentration. Figure 9 is a graph showing the relationship between the amount of water replaced and the peak height when analysis was performed using the apparatus shown in Figure 1. Figure 10 is a graph showing the relationship between the amount of water replaced and the peak height. Figure 1 shows the THF concentration in dilution water when analysis is performed using the apparatus shown in Figure 1 as displacement water it k 2 mt. Figure 12 is a graph showing the relationship between THF concentration in dilution water and collection efficiency in the same analysis, and Figure 13 is a graph showing the relationship between THF concentration and collection efficiency in the same analysis when the size of the concentration column was changed. A graph showing the relationship between THF concentration and peak height for dilution water, and FIG. 14 is a graph showing the relationship between THF concentration and collection efficiency for dilution water in the same case. 6... Organic solvent-based GPC column ( 1st column), 20
...Concentration column, 25...Reverse phase partition type ODS column (second column).

Claims (1)

[Claims] 1. In liquid chromatography in which components are separated by connecting a first column and a second column with different separation modes, a concentrating column is interposed between the first column and the second column. At the same time, the eluate from the first column is mixed with a solvent that will concentrate the components in this eluate into the concentration column, and this mixture is introduced into the concentration column to concentrate the components into the concentration column. After that, the concentrated components in the concentration column are eluted into the concentration column, and the concentrated components in the concentration column are introduced into the second column through a solution that acts as an eluent for the second column. Chromatography. 2. The first column is an organic solvent-based GPC column, and the second column is an organic solvent-based GPC column.
The liquid chromatography according to claim 1, wherein the column is a reversed phase partition type ODS column. 3. The liquid chromatography according to claim 2, wherein water is mixed with the eluate from the first column and the mixture is introduced into the concentration column.