JPH0244014B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method and apparatus for measuring stable isotopes. Stable isotopes are isotopes that do not have radioactivity. Since the natural abundance ratio of stable isotopes for a certain element is approximately constant, concentrated stable isotopes can be used as tracers. Compared to the radioactive isotope tracer method, the stable isotope tracer method poses no risk of radiation damage, does not require special facilities, and allows for free experimentation without any special restrictions on its use. It has its own characteristics.
However, when it comes to isotope measurements, radioactive isotope tracers only need to detect radiation, so the chemical form of the sample is often not an issue. While stable isotope tracers do not emit radiation, the chemical form of the sample is a problem, and their specificity is not very pronounced, making it difficult to perform high-sensitivity and rapid measurements. However, the current situation is that it is virtually impossible to calculate the balance of isotopes in many cases. For this reason, radioactive isotope tracers come in many types of radioactivity measurement devices and are often used in biological, medical, and other fields, whereas stable isotope tracers can only be used with special mass spectrometers. , emission spectrometers, infrared spectroscopy devices, nuclear magnetic resonance devices, etc., are expensive, and measurement requires careful preparation and skill, so there are many expectations from various fields such as biology, medicine, and pharmacy. However, it has not reached the point where it is widely used. Recent developments in instrumental analysis have reinvigorated the use of stable isotope tracers, which had been stagnant for a while, and are producing concrete results in various fields. However, the measurement equipment used in the stable isotope tracer method is still of limited variety and expensive, and furthermore, careful attention and skill are required for this measurement.
The stable isotope tracer method is overwhelmingly carried out using a gas chromatograph/mass spectrometer, but the sensitivity of the mass spectrometer varies depending on the substance and is easily affected by impurities, so careful attention is required. For quantitative analysis, it is necessary to prepare a calibration curve using an internal standard substance for each substance. However, in metabolic research and the like, it is almost impossible to create a calibration curve for each substance, making it virtually impossible to obtain a quantitative balance. Nowadays, stable isotope-labeled compounds are becoming more readily available, and more than ever before there is hope for the development of new, inexpensive, and easy-to-use measurement techniques that meet research objectives. An object of the present invention is to provide a rapid, simple, and highly sensitive method for measuring stable isotope tracers using microwave spectroscopy. An object of the present invention is to provide a rapid, simple, highly sensitive, and economical isotope tracer measuring device using a microwave spectrometer as a detector. The above objects of the present invention were achieved by taking advantage of the phenomenon that molecules labeled with different isotopes give their microwave absorption spectra at completely different frequencies. Embodiments of the present invention will be described below based on the drawings. FIG. 1a is a schematic diagram of an apparatus for carrying out the method according to the invention, consisting of a sample processing apparatus 20 and a microwave spectrometer 30. The sample pretreatment device 20 is a device for guiding a sample into compounds suitable for measurement by microwave spectroscopy. The sample material is mainly subjected to oxidative decomposition or hydrogenolysis using this pretreatment device 20 . It is most preferable that the reaction is carried out continuously in the presence of a catalyst, and that the desired product is quantitatively converted by the reaction, but the reaction does not necessarily have to be quantitative, and a discharge reaction or the like may be used. A reaction example is shown below.

【table】 ~~~
oxidative decomposition
If the sample does not need to be specially separated, the sample is directly introduced into the sample pretreatment device 20. FIG. 1b is a schematic diagram of an apparatus for implementing the method according to the invention, including a sample separation device 10 and a sample pretreatment device 20.
and a microwave spectrometer 30. The sample separation device 10 is a gas chromatograph device,
A liquid chromatography device may be used, but if the sample has volatilization, a gas chromatography device is mainly used.If the sample needs to be separated in a solution state, a liquid chromatography device is used to separate the sample and the solvent is removed. Send the sample to the pretreatment device via the separation device. In addition, if the sample is nonvolatile and insoluble, such as animal or plant tissue, or if separation is not particularly required, the sample is burned, the combustion gas is introduced into a gas chromatograph, and the gas components are separated and quantified. , and may be sent to a pre-processing device. The microwave spectrometer 30 provides microwave absorption spectra for molecules labeled with different isotopes at completely different frequencies, and any type of microwave spectrometer can be used, but here we use an empty one. Using a microwave cavity spectrometer (microwave cavity gas monitor) using a solid-state microwave oscillation element with a body resonator as a sample cell,
This will be explained using ¹⁵ N tracer measurement as an example. The microwave cavity gas monitor was published in Japanese Patent Application Laid-Open No. 1986-
100887 (referred to as a transmission cavity type), or the one described as the first embodiment in Japanese Patent Application No. 56-81127 (referred to as a double modulation reflection cavity type) can be used. Figure 2 shows a transmission cavity type monitor, with a rectangular Schutarch cavity resonator 3.
1, a microwave oscillator 32, an accompanying power supply 33, a modulator 34, a lock-in amplifier 35, a termination member 36, and a recorder 37. It is configured. The operation of such monitors is well known. Using a transmission cavity type monitor as shown in Figure 2.
¹⁵ If N is to be monitored, sample separation device 1
^{The 15} N separated by 0 is converted into the form of ¹⁵ NH ₃ by the pretreatment device 20. This monitor is set by adjusting the termination member 36 of the rectangular Schüttarch cavity resonator 31 so as to continuously monitor the peak value of the absorption line of ¹⁵ NH ₃ at, for example, 22789 MHz. Alternatively, as shown in FIG. 3, ¹⁵ NH ₃ may be continuously monitored using a dual modulation reflective cavity monitor. The dual modulation reflective cavity monitor includes a rectangular Schutarch cavity resonator 31', a microwave source 32', a microwave bridge 39, an automatic frequency controller (AFC) 40, a dual modulator 34', a lock-in amplifier 35, and a sample-and-hold integral. Vessel 4
1, trigger circuit 42, amplifier 43, recorder 37
Its operation is well known. The sensitivity of this monitor is several tens of times higher than that of a transmission cavity type monitor. The example in Figure 2 or Figure 3 shows the chromatographic distillate.
Only ¹⁵ N is continuously monitored. As mentioned above, the extremely important feature of the present invention shown in these Examples is that the target stable isotope tracer in a series of chromatographic distillate fractions is isolated from a single substance (e.g.
After converting ¹⁵ N and ¹⁴ N to NH ₃ ), this substance (NH ₃ ) is continuously monitored using a microwave spectrometer with extremely high selectivity and isotope discrimination ability. High detection sensitivity and stability can be obtained. Examples of combining a gas chromatograph and a microwave spectrometer have been reported before the present invention. In general, microwave spectrometers can obtain high-resolution spectra of any polar molecules that can be vaporized, but because the absorption frequencies vary greatly depending on the compound, for example, the gas chromatograph mentioned above When directly connected to a microwave spectrometer, monitoring is performed at the frequency of the target component determined in advance. For this reason, the components that can be detected are limited to a single component. Therefore, in this case, the microwave spectrometer is effective in being able to distinguish the target component even when the chromatographic peak of that component overlaps with other components and cannot be separated, but on the other hand, the microwave spectrometer is completely insensitive to other components. from,
The overall performance of the device is the same as that of a gas chromatography detector plus an auxiliary detector for single component detection. Furthermore, it is completely impossible to continuously detect isotopes in each component that is distilled out one after another as in the present invention. It cannot be said that the conventional method, that is, an apparatus in which a microwave spectrometer is directly connected to a gas chromatograph, takes full advantage of the features of the microwave spectrometer. This is because if you want to take advantage of the high resolution of a microwave spectrometer, which is not affected by coexisting components, there is no need to perform separation using a gas chromatograph, and if you use a gas chromatograph, you can detect a single component. This is because it is often virtually meaningless to perform additional operations. In this way, an apparatus that directly connects a gas chromatograph and a microwave spectrometer and the present invention may seem similar at first glance due to the structure of the apparatus. This invention is completely different in character from the present invention, which is provided with a pre-processing device, as will be explained below. In other words, the feature of the present invention lies in the separation and detection of isotopes of specific atoms (e.g.
¹⁴ N or ¹⁵ N). Therefore, no matter what kind of molecule the chromatographic fraction is, the target atoms contained in each of the chromatographic fractions that are distilled out one after another are selected from a single molecule that can be expected to have the highest sensitivity for microwave spectroscopy (for example,
NH ₃ ) and then use the high selectivity and excellent isotope discrimination capabilities of microwave spectroscopy. Therefore, the features of the device are: firstly, it uses a microwave spectrometer with high isotope discrimination ability as a detector for stable isotope tracers, and secondly, it uses a microwave absorption signal of isotopes with high sensitivity. Another advantage is that it is equipped with a pretreatment device that can convert any sample into a common specific molecule since it differs from a wide range of compounds. Note that the sample separation device upstream of the pretreatment device may be omitted in some cases.
In addition, in the case of samples such as animal or plant tissues for which a normal separation device is used, the sample is decomposed before being introduced into the separation device, and the decomposed gas is introduced into a gas chromatograph device and continuously separated and fixed. Weigh,
Furthermore, as described above, it is possible to introduce it into a pretreatment device, convert the chemical form if necessary, and then measure it using a microwave spectrometer. Further, in the method according to the present invention, quantitative balance can be determined without creating a calibration curve for each substance because the chemical forms are unified by the sample pretreatment device. Next, taking a tracer experiment using ¹⁵ N as an example, the case where ¹⁴ N and ¹⁵ N are measured simultaneously will be explained based on FIGS. 1b and 4. In the example shown in Figure 4, ¹⁵ N of the chromatographic distillate is simultaneously
This is an example of a configuration that can also monitor ¹⁴ N.
It is a device that has the feature of easily achieving quantitative balance. In Fig. 4, ^{the 14} measurement system uses the transmission cavity type monitor shown in Fig. 2, and ¹⁴ NH ₃ e.g.
Set to continuously monitor the peak value of the 23870MHz absorption line. ^{The 15} N measurement system uses the dual modulation reflective cavity type monitor shown in FIG. 3, and continuously monitors ¹⁵ NH ₃ using, for example, the 22789 MHz absorption line as described above. In FIG. 4, the transmission cavity monitor of FIG. 2 is used as the ¹⁴ N measurement system, but the dual modulation reflection cavity monitor of FIG. 3 may of course be used instead. If the sample can be vaporized, a gas chromatograph is used as the sample separation device 10. The sample separated into each component by the gas chromatography device is then sent to a reaction tube, which is a pretreatment device 20 filled with a Ni catalyst, and undergoes a hydrogenolysis reaction while mixing with hydrogen, so that ¹⁵ N becomes ¹⁵ N ₃ Here, the decomposed gas enters the two microwave spectrometers 30,
¹⁵ NH ₃ and ¹⁴ NH ₃ are detected and quantified, respectively. Therefore, the total amount of each component out of ¹⁵ can be calculated and the desired quantitative balance can be obtained. As mentioned before, if the sample is non-volatile or insoluble, such as animal or plant tissue, the sample will be decomposed and the decomposed gas will be introduced into the gas chromatograph. Nitrogen automatic analyzer
With the Model 185, operations are performed quickly in a series. Here, N ₂ in the combustion gas is detected by a gas chromatograph detector, and subsequently N ₂ is reduced to NH ₃ under an ammonia synthesis catalyst, which is measured by a microwave spectrometer. Although the reaction of converting N ₂ to NH ₃ is not a quantitative reaction, the total amount of N ₂ is determined using a gas chromatograph detector, and a microwave spectrometer determines the amount of ¹⁵ NH ₃ , ¹⁴ NH ₃
As a result, the desired quantitative balance can be obtained regardless of the chemical form of the sample. . As described above, in the present invention, unifying the chemical forms using the sample pretreatment device has an extremely important meaning in order to make it possible to maintain a quantitative balance. On the other hand, if a sample pretreatment device was added to the gas chromatograph-mass spectrometer, it would be possible in principle to achieve the same quantitative balance as the present invention, but in this case, good results would be achieved. I can't expect that. For example, in a tracer experiment using ¹⁵ N, the sample is hydrogenolyzed and
When converted to NH ₃ , the mass number is 17 ( ¹⁴ NH ₃ ) and
We will focus on the signal of 18 ( ¹⁵ NH ₃ ).
In this case, the correct value cannot be determined because the mass spectrometer cannot substantially distinguish the signal from H ₂ O. Further, if a high-resolution mass spectrometer is used at this time, this discrimination becomes possible, but the reality is that it cannot be used for tracer experiments due to its low sensitivity. Furthermore, even if you attempt to oxidize and decompose a sample and measure it with N ₂ , accurate values cannot be expected because the mass spectrometer is highly vacuumed and is easily affected by air. In addition, if the cracked gas produced by the pretreatment is insufficiently purified, interference with various fragments occurs, making it impossible to achieve the desired quantitative balance. In contrast, in the method according to the present invention, the microwave spectrometer is not affected by coexisting components, so the above-mentioned drawbacks are completely overcome. Furthermore, in mass spectrometry, even if the sample is sufficiently purified, discrimination of isobaric substances still makes tracer experiments difficult. For example, in a ¹³ C tracer experiment, 1.181% ( ¹³ C; 1.07%, ¹⁷ O; 2×
It is said that significant detection of ¹³ C is difficult in a system that contains CO ₂ with a mass of 45 (0.037) and is diluted 10 ⁴ times. Moreover, for substances with large molecular weights, the proportion of isobaric substances increases accordingly.
Furthermore, isotope discrimination becomes difficult. Techniques such as the use of double-labeled compounds have been devised to avoid this problem, but these techniques are complicated and not common.
Other measurement methods used in the stable isotope tracer method include emission spectroscopy for ¹⁵ N, and infrared spectroscopy and nuclear magnetic resonance for ¹³ CO ₂ , but all of them require batch-type measurements. It is a method that requires a great deal of effort and careful preparation and skill, and is far from a quick, easy, and highly sensitive measurement method, and it is generally impossible to obtain a quantitative balance. On the other hand, this measurement method has no problems such as isobarism, is unaffected by coexisting components, is easy to operate, and is a breakthrough that allows multiple pieces of information to be obtained continuously and quantitative balance can be calculated. This is a standard measurement method. Furthermore, as mentioned earlier, one of the features of the present invention is that when a dual modulation reflection barrel type microwave monitor or a transmission cavity type microwave monitor is used as a microwave spectrometer, it is possible to detect isotopes. The reason is that it has high sensitivity for detection. Taking the measurement of ¹⁵ NH ₃ and ¹⁴ NH ₃ in a ¹⁵ N tracer experiment as an example, a transmission cavity microwave monitor
The detection sensitivity of ¹⁵ NH ₃ and ¹⁴ NH ₃ is 0.08 ppm (S/N ~
1), and the sensitivity of the dual modulation reflective cavity microwave monitor has been improved by several tens of times.
The detection sensitivity is about 10ppb. The internal volume of these microwave cavity sample cells is approximately 60 ml, and when calculating the weight of NH ₃ in the sample cell with a sample pressure of approximately 5 Torr, the detection limit is 10 ^-11 to ^{10 -12} , which is an extremely sensitive analysis method. I understand that there is something. In addition, since the apparatus of the present invention does not use large electromagnets or delicate electrical equipment, it is characterized by being extremely simple and economical compared to gas chromatography mass spectrometry and the like.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an apparatus for implementing the method of the present invention, FIG. 2 is a diagram showing an example of the microwave spectrometer in FIG. 1, and FIG. 3 is a diagram showing another example of the microwave spectrometer. The figure shown in FIG. 4 is a diagram showing still another example of a microwave spectrometer. 10...Sample separation device, 20...Sample pretreatment device, 30...Microwave spectrometer, 31, 31'...
...Schutarch cavity resonator, 32, 32'...Microwave source, 33...Power source, 34, 34'...Modulator, 35...Lock-in amplifier, 36...Terminal member, 37, 37'...Recorder , 38...terminal circuit,
39...Microwave Bridge, 40...AFC,
41...differential sample hold integrator, 42...
Trigger circuit, 43...amplifier.

Claims (1)

[Claims] 1. Divide a sample containing a stable isotope, convert the separated sample into a chemical form containing the stable isotope that has a chemical form suitable for microwave spectroscopy, and detect the microwave absorption line by this compound. A stable isotope measurement method characterized by measuring the peak value of by microwave spectroscopy. 2. A sample separation device that separates a sample containing a stable isotope, a sample pretreatment device that converts the separated sample into a compound containing the stable isotope that has a chemical form suitable for microwave spectroscopy, and a sample pretreatment device that separates a sample containing a stable isotope. A stable isotope measurement device consisting of a microwave spectrometer for quantifying compounds created by a processing device by absorption of microwave energy by the compounds. 3. The measuring device according to claim 2, wherein the sample is a volatile sample, and the sample separation device is a gas chromatograph device. 4. The measurement device according to claim 2, wherein the sample is a solution and the sample separation device is a liquid chromatography device. 5. The measuring device according to claim 2, wherein the sample is nonvolatile and insoluble, and the sample separation device comprises a combustion device for burning the sample and a gas chromatograph device. 6. Claim 2, wherein the microwave spectrometer is at least one microwave cavity spectrometer.
The measuring device described in Items 1 to 5. 7. The measuring device according to claim 6, wherein the microwave spectrometer comprises two or more microwave cavity spectrometers each having a different resonance frequency.