JP2001059828A - Liquid chromatography nmr method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable measurement even in the case of a sample in low concentration by controlling the stop and flow measurement or the fraction loop measurement of a same mixture sample by the elution time information of a sample component read from an MS chromatogram. SOLUTION: Prescribed high frequency magnetic field pulse is irradiated with a magnet 5 for the partial image of a sample eluted successively from a liquid chromatograph 1 to observe an FID signal, which is Fourier-transformed to obtain the NMR spectrum of the partial image of each sample. The MS chromatogram of sample solution eluted previously by a mass analyzer 10 is prepared, and stop and flow measurement and fraction loop measurement are controlled on a same mixture sample on the basis of the information of the elution component of each sample component read therefrom. LC-NMR measurement for the sample component in low concentration can be performed even by the NMR device low in detection sensitivity by performing on the MS chromatogram detected by the mass analyzer 10 high in the detection sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid chromatograph-NMR method, and more particularly to a liquid chromatograph-NMR method which cannot detect a U-ray which cannot be detected by a UV detector.
The present invention relates to a liquid chromatograph-NMR method capable of easily preparing a chromatogram of a sample component having no V absorption, and suitably performing a stop & flow measurement and a fraction loop measurement of NMR based on the chromatogram.

[0002]

2. Description of the Related Art FIG. 1 shows a liquid chromatograph (L)
1 shows a configuration of an LC-NMR apparatus for analyzing a sample component eluted from C) using an NMR apparatus. 1 in the figure
Is a high performance liquid chromatograph (HPLC: high p
2 is a personal computer (PC) for controlling HPLC. H
The PLC 1 aspirates the solvent from the solvent reservoir 3 by a built-in pump (not shown) and also sucks a sample solution from the built-in sample reservoir (not shown), and develops the sample components for each component in a built-in column (not shown). ·To separate.

Many of the developed and separated sample components exhibit absorption in the UV wavelength band, and are sequentially detected by the UV detector 4 when passing through the UV detector 4 to give a UV chromatogram. After that, the sample was NMR magnet 5
Is introduced into the NMR probe 6 inserted in the sample, and used for measurement of an NMR spectrum. This NMR measurement is performed in synchronization with the detection signal from the UV detector 4, that is, the UV chromatogram (however, a delay time (time / time) until the sample component detected by the UV detector 4 moves to the NMR probe 5. Lag) is performed)
An NMR spectrum for each sample component separated in step 1 can be obtained. The NMR measurement at this time is generally performed by irradiating the sample with a predetermined high-frequency magnetic field pulse and observing the FID derived from the sample component while eliminating unnecessary signals derived from the solvent, and is mainly performed by the spectrometer 7. , The spectrometer 7 is controlled by a system 8. Processing and analysis of the acquired NMR data is mainly performed by the system 8.

[0004]

In such a configuration, the NM of the sample component separated by HPLC 1
If the R measurement is performed with high sensitivity, the detection sensitivity of NMR is not good. Therefore, when the sample components reach the NMR probe 6 by the stop & flow method, the liquid sending is temporarily stopped and the integrated measurement of NMR is performed. Set, fraction
By switching the flow path of the sample solution by the loop method, a part of the sample solution is stored in the fraction loop, and after the separation by HPLC 1, each component is sequentially sent from the fraction loop to the NMR probe 6 to perform integrated measurement of NMR. By doing so, it is widely practiced to increase the measurement time and increase the NMR detection sensitivity.

However, in order to perform the stop-and-flow method or the fraction loop method, sample components must be separated by HPLC.
The exact time of elution from 1 (retention time of sample components by HPLC) must be known. So, conventionally,
The detection signal (UV chromatogram) from the UV detector 4 has been used for this purpose. However, HP
Not all sample components eluted from LC1 have an absorption in the UV wavelength band, and some sample components do not have UV absorption. In such a case, the detection by the UV detector 4 cannot be performed, so that the NMR apparatus itself has been used as a detector instead of the UV detector 4.

The method of using the NMR apparatus itself as a detector is divided into a preliminary experiment and a main experiment.
The sample solution eluted from No. 1 is subjected to NMR measurement. That is, as a preliminary experiment, HPL
The eluate from C1 is continuously introduced into the NMR probe 6 by an on-flow method, and the sample is generally irradiated with a predetermined high-frequency magnetic field pulse to eliminate unnecessary signals derived from the solvent while removing FID derived from the sample component. The exact time at which the target sample component elutes from the HPLC 1 (retention time of the sample component by HPLC (retention
Time)) is determined as the time at which the NMR signal appears, and an NMR chromatogram of the sample solution is obtained. Next, as the present experiment, based on the NMR chromatogram obtained in the preliminary experiment, the integrated measurement of NMR using the stop & flow method or the fraction loop method is performed.

The advantage of this method is that the time required for elution from the HPLC 1 can be effectively determined even for sample components having no UV absorption. However, at present, this method has a major problem in terms of detection sensitivity. As is clear from the example of a 500 MHz NMR apparatus, ¹ H-N
In the case of MR, the range of several μg to several tens μg is the lower limit for detecting a sample. In the case of a sample smaller than this, the NMR signal is too weak and the stop & flow method or the fraction loop method is used. N that is strong enough to
The inability to obtain an MR chromatogram.

In view of the above, an object of the present invention is to provide a UV
Even for samples without absorption and low concentration, NM
An object of the present invention is to provide an LC-NMR method capable of performing a stop & flow method or a fraction loop method of R.

[0009]

In order to achieve this object, an LC-NMR method according to the present invention is directed to FID by irradiating a sample fraction sequentially eluted from a liquid chromatograph apparatus with a predetermined high-frequency magnetic field pulse. And observe the observed F
In the liquid chromatography-NMR method of obtaining the NMR spectrum of each sample fraction by Fourier transforming the ID, an MS chromatogram of a sample solution eluted from the liquid chromatograph using a mass spectrometer is prepared in advance,
Based on the information on the elution time of each sample component read from the MS chromatogram, control for stop and flow measurement or fraction loop measurement of liquid chromatography-NMR for the same mixture sample was performed. It is characterized by:

The sample fractions eluted sequentially from the liquid chromatograph are irradiated with a predetermined high-frequency magnetic field pulse to observe the FID, and the observed FID is Fourier-transformed to obtain an NMR spectrum of each sample fraction. In the liquid chromatography-NMR method to be obtained, a part of the sample solution introduced from the liquid chromatograph into the NMR apparatus is fractionated and introduced into the mass spectrometer, and the MS of the sample solution is analyzed using the mass spectrometer. A chromatogram is created, and based on the information on the elution time of each sample component read from the MS chromatogram, control for liquid chromatography-NMR stop & flow measurement or fraction loop measurement is performed. It is characterized by doing.

Also, by transferring information on the retention time of the sample component read from the MS chromatogram to the infrared spectrometer, the infrared spectrum of the sample component separated by the liquid chromatograph can be measured. It is characterized by doing so.

[0012]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 2 shows the LC according to the present invention.
1 shows an embodiment of an NMR apparatus. 1 in the figure
Is a PC that controls HPLC1 and HPLC is a PC that controls HPLC1.
The HPLC 1 was equipped with a built-in pump
, And a sample solution is also sucked from a built-in sample reservoir (not shown), and sample components are developed and separated for each component by a built-in column (not shown).

The sample components developed and separated for each component are U
Since many of them exhibit absorption in the V wavelength band, they are sequentially detected by the UV detector 4 when passing through the UV detector 4. Thereafter, the sample solution was mixed with N 2 inserted into the NMR magnet 5.
It is introduced into the MR probe 6 and used for NMR spectrum measurement. This NMR measurement is performed in synchronization with a detection signal from the UV detector 4, that is, a UV chromatogram (however, a delay time (time / time) until a sample component detected by the UV detector 4 moves to the NMR probe 6. By carrying out (lag), it is possible to obtain an NMR spectrum for each sample component separated by HPLC1. The NMR measurement at this time is generally performed by irradiating the sample with a predetermined high-frequency magnetic field pulse and observing the FID derived from the sample component while eliminating unnecessary signals derived from the solvent, and is mainly performed by the spectrometer 7. , The spectrometer 7 is controlled by a system 8.

On the other hand, after the UV detector 4, an HPLC
A flow path switch 9 for switching the flow path of the sample solution eluted from 1 is provided, so that the flow path of the sample solution introduced into the NMR probe 6 can be switched to the mass spectrometer 10 side. . By switching the flow path switch 9 from the NMR probe 6 side to the mass spectrometer 10 side, sample components eluted from the HPLC 1 can be sequentially mass analyzed.

Now, by switching the flow path from the HPLC 1 to the mass spectrometer 10 and displaying the change in the total amount of ions detected by the mass spectrometer 10 as a function of time, the total ion chromatogram (TIC: Total Io
n Chromatogram). If the change in the amount of only ions having a specific mass-to-charge ratio is displayed as a function of time, the partial ion chromatogram (RIC:
Regional Ion Chromatogram). As shown in FIG. 3, these are all widely known as a kind of MS chromatogram.

By the way, in such a configuration, the HP
When there is a component having no UV absorption among the sample components eluted from LC1, an NMR spectrum of all components is acquired by the stop & flow method or the fraction loop method synchronized with the UV chromatogram. It is not possible. When the concentration of the sample component eluted from the HPLC 1 is lower than a predetermined value, an NMR chromatogram is obtained by using an NMR apparatus as a detector as in the conventional method, and the sample component in the NMR chromatogram is obtained. It is also difficult to perform a stop-and-flow method or a fraction loop method based on the elution time of NMR because of insufficient sensitivity of the NMR apparatus.

Therefore, in such a case, the measurement is to be performed in two stages, that is, a preliminary experiment and a main experiment. First, as a preliminary experiment, the flow path switch 9 is switched to the mass spectrometer 10 side and turned on. -The MS chromatogram of the sample component eluted from HPLC1 is measured by the flow method. The MS chromatogram obtained at this time is, as shown in FIG. 4A, an MS spectrum of the sample component developed along the axis of the retention time (retention time) of HPLC. Then, the ion intensity of each MS spectrum is calculated as m /
When integrating on the z-axis and projecting the result on the time axis, TIC
(Total ion chromatogram) can be obtained. The scale of the TIC obtained in this manner is referred to as HP
When displayed as the retention time (retention time) of LC1 ((b) of FIG. 4), it becomes an MS chromatogram of the sample component eluted from HPLC1.

Based on the information on the retention time (retention time) of the MS chromatogram, the elution peak (eg, a) of the sample component to be subjected to NMR measurement is designated as a region (FIG. 4 (c)), and the peak N according to elution time
HPLC for stop & flow measurement of MR
Automatic input of time conditions from the system 12 controlling the mass spectrometer 10 to the PC 2 controlling the mass spectrometer 10 via the cable 11. At this time, if necessary, a delay time (time / time) for correcting a time lag based on a difference between the flow path on the mass spectrometer 10 side and the flow path on the NMR probe 6 side.
(Lag) is also input.

When it is desired to perform a fraction loop measurement instead of a stop & flow measurement, a fraction loop loop (not shown in FIG. 2) is provided between the flow path switch 9 and the NMR probe 6. At the same time as the unit is inserted, information on the retention time (retention time) in the MS chromatogram is input from the system 12 to the fraction loop unit so that the time of switching the flow path of the fraction loop is controlled. . At this time, if necessary, a delay time (time lag) for correcting a time lag based on a difference between the flow path on the mass spectrometer 10 side and the flow path on the fraction loop unit side is also input. To do.

When the conditions of the PC 2 controlling the HPLC 1 or the fraction loop unit (not shown) are set in this way, the flow path switch 9 is moved from the mass spectrometer 10 to the NMR probe 6. Switch to
The NMR measurement of this experiment is started. As in this example, MS acquired by the mass spectrometer 10 having high detection sensitivity
If a stop-and-flow method or a fraction loop method based on a chromatogram is adopted, even an NMR apparatus with low detection sensitivity can perform LC-NMR measurement on a low-concentration sample component.

In this example, an NMR based on TIC is used.
Although the measurement was described, as shown in FIG. 3, NM (partial ion chromatogram) was used instead of TIC based on NM.
If PC2 or a fraction loop unit (not shown) is set for time to perform R measurement, only a sample component that gives an ion having a predetermined mass-to-charge ratio (or a specific substituent) is targeted. It is also possible to perform a stop & flow method or a fraction loop method of NMR measurement.

In this embodiment, the flow path switching device 9 is provided at the subsequent stage of the UV detector 4.
It may be provided before the detector 4.

Next, FIG. 5 shows another embodiment according to the present invention. In FIG. 5, a solvent 13, a pump 14, a sampling device 15, a column 16, and a UV detector 4 all represent a general device configuration of an HPLC device. The arrows in the figure indicate the flow of the liquid (the solvent or the solvent containing the sample components).

In such a configuration, the eluate of the HPLC exiting the UV detector 4 is separated by the splitter 17 into two eluates.
One is introduced into the mass spectrometer 10,
The rest is directly NM when performing stop & flow measurement
When the fraction loop measurement is performed on the R device 18, the fraction loop unit 19 is introduced into the NMR device 18 after passing through the fraction loop unit 19. At this time, the branching ratio of the splitter 17 is set so as to be, for example, 1:20 in consideration of a difference in detection sensitivity between the mass spectrometer 10 and the NMR device 18. Synchronous control of these devices is performed on the computer 20.

The mass spectrometry and the NMR measurement of the HPLC eluate that has exited the UV detector 4 can be performed slightly earlier in time in the mass spectrometry than in the NMR measurement after the eluate is branched by the splitter 17. After the flow path length is selected as described above, the operations are performed almost in parallel. Therefore, in accordance with the appearance time of the peak of the TIC, which is monitored earlier by the mass spectrometer 10 which is much more sensitive than the NMR spectrometer,
It is possible to stop the pump 14 of the HPLC apparatus or switch the flow path of the fraction loop unit 19 in real time. As a result, even if the sample component in the eluate has a low concentration and does not have UV absorption, the TI
Based on C, it is possible to perform stop & flow measurement or fraction loop measurement of the eluate branched to the NMR apparatus 18 side in real time, and in the above embodiment, an MS chromatogram is obtained in advance. Preliminary experiments needed for this became unnecessary in this example,
Samples are saved.

In this example, an NMR based on TIC is used.
Although the measurement has been described, as shown in FIG. 3, if the control of the pump 14 of the HPLC apparatus and the switching of the flow path of the fraction loop unit 19 are performed based on the RIC instead of the TIC, a predetermined mass charge can be obtained. Ratio (or
The stop & flow method and the fraction loop method of the NMR measurement can be performed in real time only for the sample component that gives an ion having a specific substituent).

In this embodiment, the splitter 17 is provided at the subsequent stage of the UV detector 4.
May be provided before the UV detector 4.

Further, when information on the retention time of each sample component in the MS chromatogram obtained in this manner is passed to an analytical instrument such as a Fourier transform infrared spectrometer (FT-IR), each of the separated components by HPLC is obtained. It is also possible to acquire an infrared absorption spectrum for each sample component (LC-IR).

[0029]

As described above, the LC-NM of the present invention
According to the R method, an MS chromatogram is acquired by a mass spectrometer that is much more sensitive than an NMR device,
Based on the MS chromatogram, the timing for stopping the sample solution feed for the stop & flow method and the timing for switching the flow path of the sample solution for the fraction loop method can be set. Is low concentration and does not have UV absorption, it is possible to perform NMR stop & flow measurement or fraction loop measurement.

[Brief description of the drawings]

FIG. 1 is a diagram showing a conventional LC-NMR apparatus.

FIG. 2 is a diagram showing one embodiment of the LC-NMR apparatus of the present invention.

FIG. 3 is a diagram showing types of MS chromatograms.

FIG. 4 is a diagram showing a method for setting conditions for LC-NMR measurement from an MS chromatogram.

FIG. 5 is a view showing another embodiment of the LC-NMR apparatus of the present invention.

[Explanation of symbols]

1 ... HPLC, 2 ... PC, 3 ... Solvent reservoir, 4 ... UV detector, 5 ... Magnet, 6 ... NMR probe, 7 ...
Spectrometer, 8 ... System, 9 ... Flow path switcher 1
0: mass spectrometer, 11: cable, 12: system, 13: solvent, 14: pump, 15: sampling device, 16: column, 17: splitter , 18 ...
NMR apparatus, 19: fraction loop unit, 20: computer.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kamo Osamu 1-2-1, Musashino, Akishima-shi, Tokyo Japan Electronic Co., Ltd. (72) Kazuo Tanaka 3-1-2, Musashino, Akishima-shi, Tokyo Japan Electronics Inside the corporation

Claims (3)

[Claims] 1. A method for irradiating a sample fraction eluted from a liquid chromatograph apparatus with a predetermined high-frequency magnetic field pulse to F
In the liquid chromatography-NMR method of observing the ID and Fourier transforming the observed FID to obtain an NMR spectrum of each sample fraction, the MS of the sample solution eluted from the liquid chromatograph using a mass spectrometer in advance is used. A chromatogram is prepared, and based on the information on the elution time of each sample component read from the MS chromatogram, for liquid chromatograph-NMR stop & flow measurement or fraction loop measurement of the same mixture sample. Liquid chromatography-NMR method, characterized in that: 2. A method for irradiating a sample fraction eluted from a liquid chromatograph apparatus with a predetermined high-frequency magnetic field pulse to F
In the liquid chromatography-NMR method in which the ID is observed and the observed FID is subjected to Fourier transform to obtain an NMR spectrum of each sample fraction, a part of the sample solution introduced from the liquid chromatograph apparatus to the NMR apparatus is separated. The sample is introduced into a mass spectrometer, and an MS chromatogram of the sample solution is created using the mass spectrometer. Based on the information on the elution time of each sample component read from the MS chromatogram, the liquid A liquid chromatograph-NMR method characterized in that control for stop and flow measurement or fraction loop measurement of chromatograph-NMR is performed. 3. An infrared spectrum of a sample component separated by a liquid chromatograph device can be measured by passing information on a retention time of the sample component read from the MS chromatogram to an infrared spectrometer. The liquid chromatograph-NMR method according to claim 1 or 2, wherein