Highly selective chromatography-molecular rotational resonance spectroscopy system and method

Abstract

The capabilities of gas or liquid chromatography-molecular rotational resonance (GC / LC-MRR) instrumentation exceed those of high-resolution mass spectrometry and nuclear magnetic resonance in terms of selectivity, resolution, and compound identification. MRR detection provides high specificity for selective gas or liquid phase separations, including the ability to resolve co-eluting peaks and isomeric compounds without any loss of specificity or accuracy. MRR allows for qualitative identification and absolute quantification of analyte components separated by GC or LC in the absence of reference standards. GC-MRR is ideal for compound-specific isotope analysis (CSIA) and can identify enantiomers and enantiomeric excess. GC-MRR measurements are particularly useful for studying biosynthesis / degradation and geochemical isotopic compounds.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Application No. 62 / 977,846, filed on February 18, 2020, entitled “Molecular Rotational Resonance Spectrometer for Measurement of Gas Chromatography Effluents”; U.S. Application No. 62 / 913,082, filed on October 9, 2019, entitled “A Molecular Detection / Separations System of Singular Specificity”; and U.S. Application No. 62 / 844,280, filed on May 7, 2019, entitled “Hyphenated MRR Spectroscopy: MRR Spectroscopy as a Chromatographic Detector”. Each of these applications is incorporated herein by reference in its respective entirety. Background Art

[0003] Chromatographic separation techniques are often used to identify and quantify components in a chemical mixture. Chromatography is used for both preparative and analytical purposes. In preparative chromatography, the instrument physically separates and isolates different compounds. In analytical chromatography, the instrument identifies and quantifies different components in a sample. For example, gas chromatography (GC) is used to characterize small molecule mixtures during drug development and approval and to measure impurities in drug products. The U.S. Food and Drug Administration (FDA) relies heavily on gas chromatography for regulatory validation.

[0004] Conventional chromatographic equipment comprises a column, and the column comprises a stationary phase that retains the analyte in the column for different amounts of time based on the weight, polarity or other properties of different analytes. This allows these components to be separated. Conventional chromatographic equipment also comprises a detector, and when the chemical component is eluted from the column, the detector returns a signal. Some detectors in these detectors cannot obtain chemical information-for example, flame ionization detector (FID), which returns a signal due to the combustion of the analyte. Other detectors do obtain chemical specific information-for example, mass spectrometry (MS). However, for all detectors, the column should completely or almost completely separate the various components of the mixture, so as to reliably quantify the components. When more than one component is eluted from the column at the same time, most detectors cannot accurately resolve and quantify the individual components. Therefore, analytical chemists have made great efforts to develop new columns that can better separate challenging mixtures and develop methods that can completely separate individual components. Despite a lot of efforts, there are still some important analyses in which chromatographic separation is impossible or very difficult (expensive columns and long run times are required).

[0005] Molecular rotational resonance (MRR) spectroscopy (also known as molecular rotation spectroscopy or microwave spectroscopy in other contexts) characterizes compounds by their pure rotational angular momentum transitions in the gas phase. The rotational energy levels of a molecule are quantified as specified by its 3-dimensional mass distribution, which is represented by its moment of inertia I. This is defined (in one dimension) as I = Σm i r i 2 , where m i is the mass of atom i in the molecule, and r i is the distance of atom i from the center of mass of the molecule. The rotational spectrum of a molecule is described by a Hamiltonian that depends precisely on its moments of inertia about the three spatial axes, and thus using rotational spectroscopy, molecules can be unambiguously distinguished by their structural differences. Given their numerous and extremely narrow spectral lines (typical spectral resolution νΔν≈10 -5 ), high-resolution rotation spectra are therefore absolutely unique for each molecular structure.

[0006] For example, Figure 1 Shows 13 CH 3 CN and CH 3 13 MRR spectra of CN - isotopomers with the same mass, where clearly resolved spectral patterns can be observed for each spectrum. In addition, it is noteworthy that the MRR spectrum of each compound can be calculated with high accuracy. Therefore, using MRR, compounds can be unambiguously identified without reference samples. Summary of the invention

[0007] Instruments that combine gas chromatography (GC) or liquid chromatography (LC) with molecular rotational resonance (MRR) spectroscopy provide unprecedented levels of molecular information about analytes in the gas or liquid phase. The resulting GC-MRR or LC-MRR spectroscopy system (also referred to as a GC-MRR or LC-MRR instrument) can employ broadband MRR measurement techniques, including chirped pulse FT (CP-FT) techniques, to measure spectra several orders of magnitude faster than other MRR or rotational spectroscopy systems. The GC-MRR or LC-MRR instrument of the present invention has at least three advantages over other GC or LC detection systems, specifically mass spectrometry (MS): (i) MRR is highly sensitive to differences in molecular structure and can therefore resolve all types of isomeric compounds; (ii) MRR can resolve and quantify co-eluting compounds without loss of specificity or accuracy; and (iii) both qualitative identification and absolute quantification can be achieved without reference standards.

[0008] The instrument of the present invention can take the form of an integrated chromatography-spectrometric system, which includes a gas chromatograph, an MRR spectrometer, and a processor operably connected to the MRR spectrometer. In operation, the gas chromatograph separates the analyte into components using a column. The column is fluidly connected to a carrier gas source, which supplies carrier gas to push the components through the column. MRR measures the chromatogram of the components. The MRR includes a measuring chamber, a nozzle, and a microwave source. The measuring chamber holds the components of the analyte separated by the gas chromatograph. The nozzle, which is fluidly connected to the column and the measuring chamber, injects the components into the measuring chamber. And the microwave source in electromagnetic communication with the measuring chamber excites the components in the measurement with an excitation pulse, and the excitation pulse has at least one spectral component in the frequency range of about 6 GHz to about 18 GHz. The processor resolves the components of the analyte based on the MRR spectrum of the components.

[0009] An alternative instrument includes a chromatograph and an MRR spectrometer in fluid communication with the chromatograph. The chromatograph separates the analyte into components. And the MRR spectrometer measures the MRR spectrum of at least one of the components.

[0010] Another inventive instrument comprises a gas chromatograph, an MRR spectrometer, and a pulse jet expansion source in fluid communication with a column of the gas chromatograph and a vacuum chamber of the MRR spectrometer. Again, the gas chromatograph separates analytes into components, and the MRR measures the MRR spectra of the components. The pulse jet ultrasonic expansion source transports the components from the column into the vacuum chamber.

[0011] In the instrument of the present invention, the raw data collected by the MRR spectrometer is a series of continuous time-domain free induction decay (FID) traces from the analyte components separated by the chromatograph. Each of these time-domain FID traces can be Fourier transformed with a processor (e.g., an instrument processor or a separate processor) to produce a corresponding molecular rotation resonance (MRR) spectrum. The processor identifies the spectral lines of each MRR spectrum in these MRR spectra and sums the amplitudes of the spectral lines in each MRR spectrum to produce an amplitude value corresponding to a time bin (time bin) of the time-domain FID trace. The processor forms a chromatogram of the analyte components based on the amplitude values ​​and time bins of the time-domain FID traces. The processor also identifies the peaks in the chromatogram and sums, integrates or averages the time-domain FID traces corresponding to the time bins associated with the peaks in the chromatogram to produce an integrated time-domain FID trace. Fourier transforming the integrated time-domain FID trace produces an MRR spectrum associated with the peak in the chromatogram.

[0012] All combinations of the foregoing concepts and additional concepts discussed in more detail below (provided that these concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly used herein that may also appear in any disclosure incorporated by reference should be given the meaning most consistent with the specific concepts disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The skilled artisan will appreciate that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily drawn to scale; in some cases, various aspects of the inventive subject matter disclosed herein may be exaggerated or enlarged in the drawings to facilitate understanding of different features. In the drawings, like reference numerals generally refer to like features (e.g., functionally similar and / or structurally similar elements).

[0014] Figure 1 Molecular rotation resonance (MRR) spectra of two isotopomers of acetonitrile are shown, demonstrating the selectivity for isobaric compounds and the extremely high resolution of the MRR spectral lines.

[0015] Figure 2A Chlorinated dioxin isomers that have never been separated by gas chromatography (GC) but can be analyzed by GC-MRR spectroscopy are shown.

[0016] Figure 2BApproved fluorinated drugs and their possible des-F degradants that can be analyzed by GC-MRR spectroscopy are shown.

[0017] Figure 2C Shown is a chromatogram of co-eluting acetonitrile isotopic species that can be analyzed by GC-MRR spectroscopy.

[0018] Figure 3A is a schematic diagram of the GC-MRR system.

[0019] Figure 3B Is applicable to Figure 3A Photo of the MRR spectrometer of the GC-MRR system.

[0020] Figure 3C and 3D Shown is suitable for showing the use Figure 3A GC-MRR system and / or Figure 3B Graphical user interface view of the MRR spectra acquired by the MRR spectrometer.

[0021] Figure 4 An example of a pulse jet nozzle suitable for use in a GC-MRR system is shown.

[0022] Figure 5A Simulated MRR spectra of isopulegol at four different temperatures are shown, showing an increase in sensitivity and a shift to lower frequencies with decreasing rotation temperature.

[0023] Figure 5B Shows Figure 5A Close-up of the simulated MRR spectrum in the frequency range 6-18 GHz. At room temperature, this molecule would be completely undetectable at microwave frequencies, as the spectral line intensities drop below 10 –7 The detection cutoff value of the unit.

[0024] Figure 6 Shown are simulated MRR spectra of molecules with different molecular weights at a temperature of 300 K.

[0025] Fig. 7A Methods for calculating total molecular chromatograms (TMC) and extracted molecular chromatograms (EMC) from raw time-domain free induction decay (FID) traces collected by an integrated chromatography-MRR system are demonstrated.

[0026] Figure 7B Schematic diagram of TMC and EMC calculated from FID trace.

[0027] Figure 7C The TMCs of 24 isotopologues of five common organic molecules were obtained using the GC-MRR system.

[0028] Fig.7D Shows Fig. 7A The EMC of the 24 isotopologues in the TMC.

[0029] Fig. 8A Shown are the TMCs of (a) ethyl bromide, (b) thiophene, (c) 2-chlorothiophene, (d) 3-chlorothiophene, (e) 3-chloropyridine, and (f) 2-chloropyridine.

[0030] Figure 8B Shows Fig. 8A Two examples of how the natural isotopic abundances of compounds in can be readily obtained by GC-MRR EMC. The error in these ratios is ±3% RSD.

[0031] Fig. 9 A hybrid liquid chromatography (LC)-MRR spectroscopy system is shown.

[0032] Fig. 10A Chiral labels are shown, which are formed in the gas phase by combining chiral tags and enantiomers with different spectra of different diastereomers.

[0033] Fig. 10B Enantiomeric excess analysis from MRR spectroscopy is demonstrated. Measurements are performed on racemic or even pure enantiomers using chiral tags. DETAILED DESCRIPTION

[0034] Analytical Problems That Can Be Solved by GC-MRR

[0035] GC, LC and MRR spectroscopy are well-established techniques, although MRR spectroscopy is still in its early stages in terms of commercial adoption for analytical applications. However, GC-MRR and LC-MRR spectroscopy can solve problems that no other technique or combination of techniques can currently solve. It has been shown that direct MRR spectroscopy without chromatographic separation identifies components in mixtures containing up to about 10 components. However, actual samples in biochemistry, environment, petrochemicals and other application areas using GC and LC have matrices and multiple analytes at different concentrations. In addition, the MRR spectra of mixtures are highly complex, in part because the patterns of each component are intertwined with each other. When there are many species, identifying unknown components in a mixture (which is usually done by computer-driven pattern recognition) becomes very time-consuming, ultimately limiting the ability of analysts to interpret complex data. Therefore, GC-MRR or LC-MRR mass spectrometry greatly expands the complexity of samples that can be successfully analyzed by MRR spectroscopy, and enables accurate quantification of mixture components using the area of ​​chromatographic peaks in the MRR chromatogram.

[0036] At the same time, when used as a detector, MRR spectroscopy brings completely new capabilities to gas and liquid chromatography analysis. Specifically, when chromatographic separation is not possible or difficult to perform, MRR spectroscopy has particular advantages over other GC and LC detectors because MRR spectroscopy can still easily identify and quantify individual components that cannot be chromatographically separated.

[0037] The challenge of coelution or partial overlap of peaks is fundamental in separation science. Giddings made an implicit prediction using statistical overlap theory, which stated that when using a chromatographic column with a peak capacity of n, there is "no real hope" or separation of n compounds due to peak overlap. This theoretical problem has been demonstrated by the general resolution problem in chromatography, which states that no single column can separate all of the components in a complex mixture under given conditions. This statement is especially applicable to today's more complex separations involving isomeric components. While extraordinary progress has been made in GC and LC separations of isomers, and two-dimensional separation systems (e.g., GC×GC) have also been developed to increase peak capacity, the ability of MRR spectroscopy to identify and quantify partially or completely coeluting compounds is a major advance in analytical chemistry.

[0038] There are advanced mathematical methods for resolving overlapping peaks by area extraction, such as Fourier self-deconvolution, wavelets, multivariate curve resolution, and iterative curve fitting; however, most of these chemometric techniques fail in the following situations: (a) when the peaks completely overlap or (b) when experimentally measured reference spectra for each component do not exist. This is a situation where highly selective detectors, such as MRR spectroscopy, are very useful: MRR spectroscopy does not depend on resolving the stoichiometry of co-elutions. Instead, MRR spectroscopy produces a signal that is specific to the analyte, so that other molecules will not match it. In addition, MRR spectroscopy is selective enough that it can distinguish between two molecules with the same mass, for example, isotopomers, diastereomers, and enantiomers. This is something that even the best mass spectrometry detectors used for chromatography cannot do.

[0039] Figures 2A-2C Several examples are highlighted where GC-MRR and LC-MRR can solve challenging problems in separation science that cannot be addressed by GC or LC alone. Figure 2A Two isomeric tetrachlorodibenzodioxins (TCDD) are shown, which are persistent organic pollutants and human carcinogens. To date, no GC column can separate 1,2,3,8-TCDD and 1,2,3,7-TCDD. This analytical separation problem is exacerbated by the fact that current GC detectors produce indistinguishable signatures for these two analytes. However, MRR spectroscopy can produce distinct spectra for these compounds.

[0040] As a second example, fluorine is becoming increasingly important in pharmaceuticals, and resolving fluorinated compounds from their defluorinated counterparts in GC can be very challenging. However, distinguishing these compounds by rotational spectroscopy is straightforward. Figure 2B The structure of bictegravir, a recently FDA-approved drug, is shown, where GC-MRR was extremely useful in resolving fluorinated / defluorinated impurities.

[0041] As another example, Figure 2C The GC-MRR spectrum shows acetonitrile (CH 3 Chromatogram of a mixture of eight isotopologues of deuterated acetonitrile (DCN). Compound-specific isotope ratio analysis is important in many areas of research, including studies of metabolism and environmental degradation. Only deuterated acetonitrile (DACE) can be separated from a mixture of isotopologues on a chromatogram. 3 CN). Mass spectrometers can be used to distinguish isotopologues with different masses, but even the highest-resolution mass spectrometer detectors cannot distinguish between the two. 13 C-substituted isotopomers, 13 CH 3 CN vs CH 3 13 CN.

[0042] GC-MRR metabolomics studies (described in more detail below) show that different bacterial cultures in groundwater can have very different isotopic selectivities for the same organic compound. In addition, the above-mentioned chemometric methods, including powerful multivariate curve resolution methods, failed in this case due to high overlap. This study is an example of biosynthesis and degradation studies that can be performed with GC-MRR spectroscopy but not possible with other methods.

[0043] GC-MRR spectroscopy measurement types and frequency bands

[0044] GC-MRR spectroscopy systems can be configured to perform targeted measurements, broadband measurements (e.g., measurements across several spectral lines and / or bandwidths of 50 MHz, 100 MHz or higher), or both. Targeted measurements are similar to selected ion measurements in mass spectrometry, and are typically focused to capture the MRR spectrum of a specific species or set of species with good sensitivity. Broadband measurements are similar to total ion monitoring measurements in mass spectrometry, and are typically performed to characterize mixtures or unknown compounds. In MRR spectroscopy, broadband measurements can be based on chirped pulse Fourier transform techniques, which involve irradiating a sample with one or more pulses of chirped microwave or millimeter wave radiation, and detecting and Fourier transforming the FID signal emitted by the sample in response to the chirped pulses. Broadband measurements are typically performed on samples containing one or more unknown components. For more information on chirped pulse Fourier transform MRR spectroscopy, see, for example, the following U.S. patents, which are incorporated herein by reference in their respective entireties: U.S. Patent No. 9,046,462, entitled “Chirped Pulsed Frequency-Domain Comb for Spectroscopy”; U.S. Patent No. 9,921,170, entitled “Apparatus and Techniques for Fourier Transform Millimeter-Wave Spectroscopy”; and U.S. Patent No. 10,107,744, entitled “Frequency Hopping Spread Spectrum (FHSS) Fourier Transform Spectroscopy”. Wideband measurements can be made across the entire frequency band at once or over segments of the entire frequency band (e.g., 2 GHz or 4 GHz segments of the 6-18 GHz band) to reduce data rates such as those disclosed in U.S. Patent No. 8,873,043, entitled “Segmented Chirped-Pulse Fourier Transform Spectroscopy,” which is incorporated herein by reference in its entirety.

[0045] Targeted MRR measurements are typically performed when the goal is to determine whether a sample contains a specific compound or set of compounds. Instead of irradiating the sample with chirped pulses, an MRR instrument irradiates the sample with one or more narrowband (e.g., single-frequency) pulses and detects the FID signal emitted by the sample in response to these pulses. Because the measurement is targeted, the instrument can acquire, process, and store data faster than in broadband measurements, such as at rates of 2 Hz, 3 Hz, 5 Hz, 10 Hz, or greater. This measurement rate can be increased by performing a Fourier transform directly on the field programmable gate array (FPGA) that acquires the data.

[0046] Targeted MRR instruments can be operated without "species identification" capabilities because one or more excitation frequencies and the target species are known in advance. Targeted MRR measurements typically involve looking at a single line time versus intensity because the measured intensity is proportional to the species concentration. If the target species and GC / LC separation parameters are known in advance, the MRR spectrometer can be programmed in advance to target different spectral lines or frequency bands for different species. Consider quantifying a mixture of compounds A and B. If compound A leaves the column 10 seconds after the measurement has begun and has a strong resonance at 10 GHz, the MRR spectrometer can be programmed to interrogate the sample at 10 GHz with a measurement time of 10 seconds. Similarly, if compound B leaves the column 15 seconds after the measurement has begun and has a strong resonance at 6.8 GHz, the MRR spectrometer can be programmed to interrogate the sample at 6.8 GHz with a measurement time of 15 seconds. If an auxiliary detector is present at the column output, the auxiliary detector can trigger target measurements based on peak / eluting sequence (e.g., measuring the first eluent at 7 GHz, the second eluent at 9 GHz, etc.) or based on preliminary analysis performed by the auxiliary detector (e.g., if the auxiliary detector is a mass spectrometer or other device that can provide information about the composition of the eluent).

[0047] Targeted and broadband measurements can be performed at different frequency ranges, including frequency bands in the microwave region (e.g., in the 6-18 GHz band) or the millimeter wave region (e.g., 75-110 GHz, 260-290 GHz, or 520-580 GHz). The measurement type and measurement frequency band depend on the analyte and the desired information.

[0048] In addition, microwave MRR spectroscopy may be more sensitive than millimeter wave MRR spectroscopy due to differences in measurement conditions. Millimeter wave MRR spectroscopy is usually performed using a flow cell as a measurement chamber, which limits the molecular weight of the analyte component to about 120 amu. In addition, it usually takes several seconds for the gas to move through the flow cell, thereby reducing the time resolution of the GC output, which usually elutes components in less than one second. The GC carrier gas also dilutes the sample in the flow cell measurement, thereby reducing sensitivity.

[0049] As described below, microwave MRR measurements can be made through an expansion chamber fed by an ultrasonic expansion nozzle. This enables faster measurements, thereby maintaining the temporal resolution of the GC output, and enables the measurement of molecules with higher molecular weights as described below. Additionally, the same carrier gas that pushes the analyte components through the gas chromatograph can be used to push the sample into the expansion chamber, so the carrier gas does not cause additional dilution of the sample or reduce measurement sensitivity.

[0050] GC-MRR Spectroscopy System

[0051] Figure 3A and 3B A mass spectrometry-coupled GC-MRR instrument 300 is shown that can analyze difficult and / or complex mixtures in a single run. It can perform compound-specific isotope analysis (CSIA) that cannot be accomplished by any other method, including isotope fractionation analysis and quantification. For example, it can identify isomers and other compounds that cannot be separated using GC alone without a reference.

[0052] The GC-MRR instrument 300 comprises a gas chromatograph 310 coupled to an MRR spectrometer 320 having a temperature regulated flow interface 330. The gas chromatograph 310 has a carrier gas source 312 that flows a carrier gas such as helium, hydrogen, neon or argon through a column 314. The carrier gas pushes analytes, which may have many different chemical compositions, including isomers, isotopes, isotopomers and isotopologues, through the column 314 and into the MRR spectrometer 320 through the flow interface 330. Such analytes may be (periodically) siphoned out of a continuous stream or stream of gas or liquid, accumulated and volatilized (if appropriate), and injected into the column 314, such that the gas chromatograph effectively samples the continuous stream, much like an analog-to-digital converter (ADC) samples an analog signal.

[0053] Some or all of the components of the analyte propagate through the column 314 at different rates, and therefore may appear at different points in time at the end of the column 314. If these time points are separated widely enough, the components can be resolved at the output of the column 314. Other components (e.g., isomers) may co-elute, that is, other components may appear at the output of the column 314 at the same time or nearly at the same time and therefore cannot be resolved using GC alone.

[0054] The carrier gas pushes the (at least partially separated) components through the interface 330 and into the measurement chamber 324 of the MRR spectrometer so that the MRR spectrometer 320 can measure the MRR spectrum of the components. This interface 330 allows samples to be passed through the GC column 314 or directly injected into the MRR spectrometer 320 (for example, for pure compounds or simple mixtures that do not require GC separation). In other words, some samples may require GC separation, while other samples may not require GC separation. Samples that do not require GC separation can be injected directly into the MRR spectrometer (without passing through the GC), while other samples can be injected through the GC.

[0055] For example, the measurement chamber 324 can be a flow cell having at least one pair of holes that allow gas phase components and carrier gas to enter and leave. Alternatively, a pulse jet expansion nozzle, a continuous wave jet, or a buffer gas cooling cell can introduce the components into the measurement chamber 324 while rotationally cooling the components to obtain better measurement performance, as described in more detail below. The MRR spectrometer 320 measures the MRR spectrum of each component by subjecting the components to one or more excitation pulses of microwave and / or millimeter wave radiation. This excitation pulse is generated by a signal generator 321 such as an arbitrary waveform generator, a direct digital synthesizer, or a pulse pattern generator, and can be filtered, multiplied, and / or up-converted with an optional circuit system 322. The source 323 applies an excitation pulse to the molecules in the measurement chamber 324.

[0056] The interface 330 may also be coupled to (in fluid communication with) a second carrier gas source 352. The second carrier gas source 352 flows a second carrier gas to the interface 330 so as to push or propel the analyte component into the measurement chamber 324 of the MRR spectrometer. The first carrier gas and the second carrier gas may be different, for example, the first carrier gas may be helium or hydrogen, and the second carrier gas may be neon or argon, as described in more detail below.

[0057] The interface 330 may also be coupled to a (chiral) tag source 354 (in fluid communication with the tag source). Mixing the chiral tags from the chiral tag source 354 with the analyte components in the reservoir in the interface 330 causes the chiral tags to attach themselves to the different components. The chiral tags change the moment of inertia of the different enantiomers in the analyte components, which enables the enantiomers to be resolved and quantified according to the MRR spectra of the components as described below. The tag source 354 may store and supply other types of tags, including polar molecules for labeling non-polar molecules that do not have MRR spectra to produce complexes that have a dipole moment and can therefore be detected by MRR.

[0058] The excited molecules emit coherent radiation for several microseconds by free induction decay (FID) at their characteristic rotation frequency in response to the excitation. Receiver 326 detects the analog FID signal digitized by ADC 327. Processor 340 (e.g., field programmable gate array (FPGA)) records the digitized FID signal in the time domain and Fourier transforms it to the frequency domain to produce an MRR spectrum of the component. Using the MRR spectral library, processor 340 can identify and automatically quantify individual components of the analyte based on the MRR spectrum. Unassigned peaks in the MRR spectrum can be further analyzed for characterization using theoretical predictions of related species.

[0059] Current MRR instruments measure volatile liquids where the sample composition does not change significantly over time. Although the pulse jet source in a microwave MRR instrument typically operates at a repetition rate of 10 Hz, the instrument signal is signal averaged over a single spectral line for several seconds to minutes to record the spectrum. On the other hand, for GC-MRR spectra, the peak shapes are much narrower (seconds or even sub-seconds), so the instrument 300 records data with fine time resolution (e.g., at a sampling rate of 5-10 Hz) due to a digitizer (ADC 327) that can achieve the desired throughput rate and a processor 340 that can perform data processing operations without dead time.

[0060] The processor 340 can continuously measure and record the MRR spectra of the eluate as it leaves the GC column 314. In some cases, the processor 340 records and processes all of the MRR spectra. In other cases, the processor 340 records all of the time domain data and only Fourier transforms those segments corresponding to the "interesting" output from the GC column 314 in order to save processing resources and reduce overall processing time. The processor 340 can discard unprocessed or unchecked time domain and / or Fourier domain data.

[0061] Alternatively, an auxiliary (universal) detector 350, such as a thermal conductivity detector (TCD), may trigger the MRR measurement based on the output of the GC column 314. Figure 3AAs shown in , this auxiliary detector 350 can be online (sampling the same gas flow) or separate (e.g., a FID or MS detector in which the analyte is destroyed). When the auxiliary detector 350 detects a peak in the GC output, the auxiliary detector sends a trigger signal to the processor 340, which in turn triggers the signal generator 321 to emit an excitation pulse and measure and analyze the resulting FID signal. The processor 340 can also record all MRR data, as described above, and discard MRR data that is not mapped to a chromatographic peak sensed by the auxiliary detector 350. In addition, the auxiliary detector data can be combined with the MRR data to provide a more complete analysis of the analyte, for example, the auxiliary detector can sense components that do not have a dipole moment, and the isotopic information from the MRR spectrometer 320 can complete the characterization of other components.

[0062] For more information on triggered MRR spectroscopy measurements, see, for example, U.S. Patent No. 10,620,138, entitled “Methods and Apparatus for Direct Multiplication Fourier Transform Millimeter Wave Spectroscopy,” which is incorporated herein by reference in its entirety.

[0063] Figure 3B is a photograph of a sample example of a measurement chamber 324 and an MRR spectrometer 320 in a GC-MRR spectroscopy system. This MRR spectrometer 320 is depicted as a microwave MRR spectrometer configured to perform target measurements. In this image, the sample inlet 332 is located on the left side of the measurement chamber 324. Two spherical mirrors 328 are located on the left and right ports, i.e., the ultrasonic expansion nozzle 334 is mounted in one port, while the other port is maintained on a vacuum of an automatically controlled translation stage 344 controlled by a processor 340. The mirror 328 brings the measurement cavity into a resonant Fabry-Perot cavity. The translation stage 344 tunes the length of the resonant cavity (and therefore the resonant frequency of the cavity) to allow measurement of the target resonance of interest. Measuring the target resonance increases the measurement sensitivity. For broadband measurements, microwave horns (not shown) are used instead of the reflectors 328. These horns can be mounted orthogonal to the target reflectors 328. One horn can be attached to the closest Figure 6 The flange 329 of the reader in the embodiment of the present invention is provided, and another speaker can be attached to the opposite side (not shown). The measurement parameters including the translation stage and the excitation frequency settings and the measurement results can be operably connected to the processor 340 ( Figure 3A ) is shown on display 342.

[0064] Figure 3C and 3D Two views of a user interface that may be displayed on display 342 are shown. Figure 3C The diagram shown in FIG. 1 shows a graph showing acetonitrile (CH 3 Because the isotopologues have similar structures, the eight isotopologues (CD 3 CN) was separated from the other isotopologues. The remaining seven species simultaneously left the GC column (i.e., the other seven species co-eluted) and could not be resolved using GC alone.

[0065] Because eight acetonitrile isotopologues have different MRR spectra, the MRR spectrometer can resolve these acetonitrile isotopologues even when they are simultaneously detached from the column. An extracted MRR chromatogram of each isotopologue can be drawn, and the peaks are integrated to provide the accurate concentration of each species. In this example, eight isotopologues are mixed in equal concentrations, and 5nL of this mixture is directly injected into the MRR spectrometer for analysis. The MRR spectrometer measures and saves the broadband MRR spectrum of the mixture within 1 second. The information recorded in the MRR spectrum contains the identification of all isotopologues in the mixture and the quantity of each isotopologue in the gas chamber.

[0066] Molecular weight and spin cooling for GC / MRR spectrometer connections

[0067] A challenge of GC / MRR spectroscopy system is the upper limit of analyte molecular weight. In a GC-MRR spectroscopy system with a flow cell operating at room temperature or higher, the upper limit of molecular weight can be about 150amu, and the sensitivity at a molecular weight of 100-150amu may be limited because the MRR spectrum of room temperature molecules tends to become extremely weak when higher than 150amu. However, a pulse jet ultrasonic expansion source, a continuous wave jet or a buffer gas cooling pool is used to rotationally cool the molecules for MRR analysis while also maintaining the molecules in the gas phase. A GC-MRR instrument with a pulse jet expansion source, a continuous wave jet or a buffer gas cooling pool is able to analyze molecules with higher molecular weights (e.g., up to 400amu or higher) due to this rotational cooling.

[0068] Figure 4 An enlarged view of a pulse jet ultrasonic nozzle 400 is shown, which is suitable for injecting an analyte into a Figure 3A and 3BThe measurement chamber of the MRR spectrometer in the GC-MRR system shown. Three gases flow into the ultrasonic expansion nozzle: a GC flow inlet 410 for analyte components from the GC column; an exhaust valve inlet 430 for rapid removal of solvents or other volatile matrix components; and an optional (additional) carrier gas inlet 430. The GC flow inlet 410 and the exhaust valve inlet 420 can be made of 1 / 16" tubing and can be heated to at least 300°C to allow direct entry into the GC column at a certain temperature. Figure 4 In the , these tubes are PEEK plastic, but the connections can alternatively be made of metal with better thermal properties. The central (1 / 4") gas connection for the carrier gas is optional and can be used for purge gas to quickly clean the sample. All three gas flows are combined in reservoir 440, which can have a volume of about 500 μL or less. Reducing the reservoir volume can reduce extra-column widening.

[0069] The combined gas stream is co-expanded with the noble carrier gas through a pinhole nozzle 450 having a diameter of approximately 1 mm. The pinhole nozzle 450 is rapidly opened and closed by a solenoid valve 452 sealed with a Teflon poppet valve. On each gas pulse (approximately 1 millisecond in duration), the analyte component travels through the pinhole and into a chamber maintained at a high vacuum (approximately 10 –6 The measurement chamber of the MRR spectrometer is located at 1000 torr. Along the way through the pinhole, the molecule undergoes multiple collisions with the carrier gas. These collisions reduce the rotational temperature of the analyte components to about 1-2K.

[0070] For example, when analyte components separated by a GC column are injected into a microwave MRR measurement chamber using a pulsed jet ultrasonic nozzle, the carrier gas (e.g., neon) pressure can be set to about +2 to about +5 pounds per square inch gauge (psig). The nozzle is pulsed at 10 Hz. At each pulse injection, the nozzle injects about 1 nmol of analyte component and 1 μmol of neon into the measurement chamber. This corresponds to about 10-15 ml / min (STP) of carrier gas passing through the GC-MRR system. This is comparable to the typical flow rate of a wide-bore GC column. Neon carrier gas is injected at the nozzle (e.g., via Figure 4 The optional carrier gas inlet 430 in the MRR measurement chamber can enhance pulse valve operation. If neon carrier gas is injected at the nozzle, the GC column can be operated with a different carrier gas (e.g., hydrogen or helium) because the neon will dominate the rotational cooling caused by the ultrasonic pulse injection of the analyte components into the microwave MRR measurement chamber. Both carrier gases can be injected into the MRR measurement chamber.

[0071] Figure 5A and 5BShown are simulated MRR spectra of isopulegol (monoterpenoid and menthol intermediate, mass 154 amu) at four molecular temperatures: room temperature (300 K; bottom), 100 K (lower middle), 20 K (upper middle), and 2 K (top). Figure 5A shows the spectrum within microwave and millimeter wave frequencies from 0 to 600 GHz, and Figure 5B Spectra in the microwave band from 0 to 18 GHz are shown. These spectra show improved measurement performance at lower temperatures, which can be achieved by injecting the GC output into the vacuum chamber of the MRR spectrometer in the GC-MRR spectroscopy system using a pulsed jet ultrasonic expansion source. Pulsed jet ultrasonic expansion using an ideal carrier gas (e.g., neon, which has excellent cooling properties) can achieve rotation temperatures between 1 K and 2 K. (One tradeoff of using an ultrasonic expansion source is that the ultrasonic expansion source has a larger vacuum chamber to allow for the expansion. However, the instrument size is comparable to that of a high-end mass spectrometer.)

[0072] Figure 5A and 5B The MRR spectra in show a number of effects caused by cooling the molecular rotation. First, the MRR spectra are significantly simplified. For each molecule, there are far fewer rotational energy levels filled, and therefore far fewer transitions. This also means that more components can be resolved in a single mixture, because each molecule has fewer spectral lines. Second, the MRR spectra become several orders of magnitude stronger (as can be seen from the Figure 5A and 5B This can result in more sensitive and / or higher signal-to-noise ratio (SNR) measurements. Without being bound by any particular theory, Figure 5A and 5B The intensity increase shown is due to the smaller number of filled vibrational states. Third, the MRR spectrum is shifted to lower frequencies. The shift of the spectrum to lower frequencies is also advantageous because higher excitation power levels can be achieved using cheaper components in the microwave region (specifically below 18 GHz, for example, 6 to 18 GHz) than in the millimeter wave region (for example, 75 GHz and higher). Almost every molecule heavier than 50 amu has a spectral intensity peak in the microwave range (6-18 GHz).

[0073] Figure 6 Simulated MRR spectra at room temperature are shown for molecules of three different sizes. In general, the MRR spectra become more complex as the size of the molecule increases because more rotational and vibrational states are populated. In the pulsed jet, the vibrational states are also cooled, so the spectra of large molecules become less complex, as described above. Measuring the MRR spectra of compounds up to 300 amu becomes much simpler, and beyond this value also becomes feasible (where the main limitation becomes getting the compound into the gas phase and keeping the gas phase there).

[0074] Total Molecular Chromatogram (TMC) and Extended Molecular Chromatogram (TMC)

[0075] Fig. 7A Demonstrates the use of Figure 3A The method 700 of measuring raw data of the integrated GC-MRR instrument 300 shown (this method 700 can also be used to analyze LC-MRR data). As described above, the GC-MRR instrument 300 obtains time domain data (702) by injecting (at least partially) separated components from the GC column 314 into the measurement chamber 324 of the MRR spectrometer, then exciting the components in the measurement chamber 324 with microwave or millimeter wave pulses, and recording the resulting time domain free induction decay (FID) signal. Over time, the instrument 300 records a series of these time domain FID traces at a rate of 5 Hz or 10 Hz, for example.

[0076] Transforming each of these raw time-domain FID traces produces a set of MRR spectra (704), each of which is associated with a different time bin in the measurement period. The amplitudes of the spectral lines (peaks) in each of these MRR spectra are identified (706) and summed (708) to produce amplitude values ​​for the time bin associated with the MRR spectrum. Plotting the amplitude values ​​as a function of time produces a total molecular chromatogram (TMC) (710). Figure 1 Likewise, TMC represents the separation of components exiting a column in a chromatograph as a function of time, where each peak corresponds to one or more components eluting from the column in that time bin.

[0077] Each peak in a TMC can span several time bins. Identifying peaks and associated time bins (712) allows the identification of corresponding raw time-domain FID traces, which can be summed or averaged (714) and then Fourier transformed to produce an MRR spectrum (716) associated with the peak. This MRR spectrum is referred to as an extracted molecular chromatogram (EMC) and can be used to identify, resolve and / or quantify analyte components (718). For example, if a component is an isomer that cannot be resolved from a TMC, the isomer can be resolved by the difference in its MRR spectrum in the EMC. Components can be identified by comparing the EMC with a previously measured EMC library (MRR spectrum) and / or a theoretical EMC.

[0078] Figure 7BThe relationship between FID data, TMC and EMC is shown. TMC is plotted as a time series where each point (amplitude value) corresponds to the sum of the spectral lines in the MRR spectrum for the corresponding time bin. The MRR spectrum is calculated by Fourier transforming the FID trace for the corresponding time bin. In some cases, multiple analyte components can contribute to a given peak. For example, in Figure 7B In the EMC, both components 1 and 2 contribute to the left peak in the TMC and cannot be resolved from the peak itself. Component 1 contributes to the amplitude values ​​of time bins 1-8, and component 2 contributes to the amplitude values ​​of time bins 6-12. For the left peak, components 1 and 2 can be resolved, identified and quantified from the EMC. The EMC is calculated by integrating or averaging the FID traces for time bins 1-12 and then Fourier transforming the integrated or averaged FID traces.

[0079] Figure 7C and 7D A single GC-MRR analysis of 24 isotopic species of five common organic molecules is shown, demonstrating the use of Fig. 7A The method was used to demonstrate the feasibility and practicality of the GC-MRR instrument. Figure 7C Shown is TMC, wherein the peak intensity of observed transitions is summed.TMC indicates five major peaks and two small shoulder peaks just before the main acetone and acetonitrile peaks (respectively at 3.2 minutes and 7.6 minutes).These small, partially resolved shoulder peaks are fully deuterated acetone (acetone-d6) and triple deuterated acetonitrile (ACN-d3), which are separated by their associated isotopologues and isotopomer chromatograms.

[0080] Each point in the TMC contains a broadband MRR spectrum that allows separation of the individual contributions of different species. The extraordinary selectivity of MRR spectra allows resolution of the TMC into 24 EMCs, Fig.7D , where each isotopically distinct compound has one EMC. In each EMC trace, the intensities of transitions at the frequency of only that isotopic species are summed. Isotope pattern matching / analysis is not required for molecular identification, as isotopes can be directly determined based on their moments of inertia. Molecules with the same mass (e.g., H 3 C-(C=O)- 13 CH 3 and H 3 13 C-(C=O)-CH 3 or 13 CH 3 CH 2 OH and CH 3 13 CH 2 OH) can be distinguished and identified from the MRR spectrum due to their different moments of inertia.

[0081] Fig. 8A The GC-MRR spectrum and Fig. 7A TMC of ethyl bromide and five heterocyclic compounds were obtained by the method described in the previous section. Figure 8B Isotopologue-specific EMCs showing the natural isotopic abundances of ethyl bromide and 2-chloropyridine are shown. Integration of the GC-MRR peak areas provides quantitative information for all species. Fig. 8A TMC or Figure 8B The ratio between the MRR peak intensities can also be used directly as an accurate determination of the isotope ratio.

[0082] Unlike mass spectrometry, isobaric compounds do not cause problems. In addition, peak (compound) co-elution does not cause signal suppression or enhancement. This is largely due to the fact that MRR spectroscopy has a very high resolution and provides a large number of spectral lines with high specificity for one compound, even if the compound is an isomer or isotopologue or isotopologue of another compound. In addition, there is no inhibitory effect, such as that present in mass spectrometry.

[0083] There are many applications where the power of GC-MRR spectroscopy is highly useful. These applications include metabolomics, natural product screening, and environmental analysis. For example, consider the following environmental degradation study.

[0084] Determination of changes in isotopic composition is used to assess biotic and abiotic responses to organic feeds / pollutants in terrestrial and natural aquatic systems. Given that nearly all organic pollutants contain multiple stable isotopic species, compound-specific isotope analysis (CSIA) and position-specific isotope analysis (PSIA) provide broadly applicable methods to study chemical reactions in complex matrices and provide a better understanding of pollutant degradation. This degradation can occur via enzymatic pathways or via photochemical or organic reactions consisting of substitution, elimination or electron transfer.

[0085] CSIA provides deeper insights into degradation pathways by precisely locating reactive atoms in a given trace contaminant molecule. Currently, these issues are addressed by isotope ratio mass spectrometry of carbon, hydrogen and nitrogen, but isotope ratio mass spectrometry of oxygen, sulfur and chlorine is difficult. Similarly, PSIA examines intramolecular isotope changes. Conventionally, PSIA (for carbon) is performed by converting functional groups offline to carbon dioxide, followed by GC-MS, or alternatively by site-specific natural isotope fractionation-nuclear magnetic resonance (SNIF-NMR). NMR is a time-consuming, low-sensitivity technique. Therefore, significant pre-concentration of trace compounds is a requirement before NMR measurements. In contrast, GC-MRR can address these challenging situations of CSIA and PSIA without the need for post-column conversion or purification.

[0086] An example of using GC-MRR to study environmental degradation involves the selective biodepletion of diluted samples of pyridine isotopologues. Although the microbial metabolism of pyridine and other organic bases has been extensively studied, to our knowledge, no isotope-specific studies exist. Table 1 shows 14 N and 15 GC-MRR results of biodepletion of N-pyridine standards in three different microbial cultures / solutions. In these cases, both pyridine isotopologues were depleted over time. 14 Selective depletion of N pyridine isotopologues, while B. cepacia and groundwater showed no significant differences. Kinetic isotope fractionation has been noted for inorganic nitrogen sources and has been used to provide insights into biological and enzymatic hydrolysis of herbicides.

[0087] Table 2 clearly shows the different kinetics of biodepletion of the various isotopologues of acetonitrile by the two different bacteria. The data show the different kinetics of biodepletion of the various isotopologues of acetonitrile by the two different bacteria. The acetonitrile of the normal species was depleted at the fastest rate by E. coli, where no differences were detectable between the three individually substituted isotopologues. Even more interesting is the fact that for V. fischeri, CH 3 13 CN isotopologues are selectively depleted. No other analytical method or combination of methods can characterize these phenomena so easily. These types of extensive studies on position-specific isotope analysis are extremely informative for biodegradation studies and are currently underexplored due to the lack of suitable analytical tools. GC-MRR addresses this lack.

[0088]

[0089] Table 1: Selective microbial depletion of different isotopes of pyridine and acetonitrile as determined by GC-MRR. The error is approximately 2.5%.

[0090]

[0091] Table 2: Selective microbial depletion of different isotopes of acetonitrile as determined by GC-MRR

[0092] body ( 13 CH 3 CN and CH 3 13 CN) is not possible by any other existing GC method.

[0093] Liquid chromatography (LC)-MRR spectroscopy

[0094] Fig. 9 A liquid chromatography (LC)-MRR spectroscopy system 900 is shown, wherein a liquid chromatograph 910 is coupled to an MRR spectrometer 920, such as a microwave or millimeter wave MRR spectrometer configured for broadband and / or targeted measurements. The liquid chromatograph 910 includes a column 914 that receives a liquid analyte, such as from a continuously flowing sample source, through an inlet 916. A solvent flowing from a solvent source 912 pushes the liquid analyte through the column 914, wherein the analyte is at least partially separated into its constituent components.

[0095] The volatilization interface 930 connects at least a portion of the separated analyte components to the MRR spectrometer 920. The volatilization interface volatilizes the analyte components, for example, the volatilization interface can heat the analyte components until they evaporate. A carrier gas such as neon or helium from a carrier gas source 934 pushes the volatilized analyte through the volatilization interface 930 and reaches a pulse jet ultrasonic nozzle 932 connected to the volatilization interface 930. The nozzle 932 injects the volatilized analyte components into the measurement chamber 924 of the MRR spectrometer, which is evacuated to a vacuum pressure (e.g., 10 –6 Similar to Figure 3A A microwave source (not shown) in the embodiment of the present invention irradiates the analyte components in the measurement chamber with one or more targeted or broadband excitation pulses. Figure 3A The receiver (not shown) in the detector detects the FID signal emitted by the volatilized analyte component in response to the excitation pulse. The electronic device (not shown) detects and processes the FID signal to produce an MRR spectrum. The MRR spectrum can be measured continuously or on demand, for example, in response to the detection of a peak in the LC column output sensed by the auxiliary detector.

[0096] Chiral Analysis by GC-MRR Spectroscopy

[0097] Chiral analysis is an area where GC-MRR can have significant added value, particularly using a gas phase complexation technique called chiral labeling to convert enantiomers into diastereomers in MRR for chiral analysis. It has been shown that small amounts of small, volatile chiral molecules effectively complex with a wide range of chemical analytes and allow accurate determination of the enantiomeric excess of these compounds within a mixture. For more information on chiral labeling, see, for example, U.S. pre-authorized publication No. 2019 / 0302015 entitled "Cavity-Enhanced Fourier Transform Spectroscopy for Chiral Analysis", which is incorporated herein by reference in its entirety.

[0098] Fig. 10A and 10B The idea behind chiral labeling is shown in Figure 1. Briefly, chiral labeling involves using a small chiral molecule (such as Fig. 10A In the pulsed ultrasonic expansion nozzle, weakly bound complexes stabilized by a combination of hydrogen bonding, van der Waals forces, and other forces can be effectively formed. The resulting diastereomeric complexes have different moments of inertia and can be separated by the above-mentioned and Fig. 10B Chiral analysis is important in a wide range of applications including pharmaceuticals, environmental analysis, and metabolite analysis.

[0099] More specifically, Fig. 10A The enantiomers of verbenone (an example chiral molecule) are shown to have identical MRR spectra because the enantiomers have identical moments of inertia. However, when a chiral tag (in this case, (S)-3-butyn-2-ol) is complexed with the two enantiomers, a difference in moment of inertia results, which produces two species with different moments of inertia and, therefore, different MRR spectra. These two complexes are referred to as heterochiral (i.e., (R,R)-verbenone + (S)-3-butyn-2-ol) or homochiral (i.e., (S,S)-verbenone + (S)-3-butyn-2-ol).

[0100] Fig. 10B Two spectral lines are shown, Fig. 10AThe spectrum of each of the heterochiral complex or homochiral complex in the sample is spectral line. In the case of the racemic mixture of the butynol label, the intensity of the two complexes is observed to be roughly equal (these two complexes are different due to complex dipole moments, formation ratios and other factors). With pure (S)-3-butyn-2-ol as the label, spectral lines of different intensities are observed. These spectral line ratios can be used to calculate the enantiomeric ratio and / or enantiomeric excess of verbenone in the sample.

[0101] Chiral monitoring can be integrated into a GC-MRR instrument with an accessory system for introducing a gas phase chiral tag through an additional valve at the interface of the GC outlet before the pulse jet sampling source. The tag can be mixed with the eluting sample post-column. The spectral library can contain entries for reference chiral molecules (chiral tags). This chiral MRR approach can be used in place of or in conjunction with chiral GC separations, opening up a range of new separation and identification capabilities.

[0102] in conclusion

[0103] Although embodiments of the present invention have been described and shown herein, a person of ordinary skill in the art will readily appreciate various other devices and / or structures for performing the functions described herein and / or obtaining these results and / or one or more of these advantages, and each of such changes and / or modifications is considered to be within the scope of the embodiments of the present invention described herein. More generally, a person of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications for which the present invention is taught. Using only routine experiments, a person of ordinary skill in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are introduced only by way of example, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner different from that specifically described and required. Embodiments of the present invention disclosed herein relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, if any combination of two or more such features, systems, articles, materials, kits and / or methods are not mutually inconsistent, then such features, systems, articles, materials, kits and / or methods are included within the inventive scope of the present disclosure.

[0104] In addition, various inventive concepts may be embodied in one or more methods for which examples have been provided. The actions performed as part of the methods may be ordered in any suitable manner. Thus, embodiments may be constructed in which the actions are performed in an order different from that shown, which may include performing some actions simultaneously, even if the actions are shown as sequential actions in the illustrative embodiments.

[0105] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0106] As used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one", unless explicitly indicated to the contrary.

[0107] As used herein in the specification and in the claims, the phrase "and / or" should be understood to mean "either one or both" of the elements so combined, i.e., elements that exist together in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally exist, whether related or unrelated to those elements specifically identified. Therefore, as a non-limiting example, when used in conjunction with open language such as "including", a reference to "A and / or B" may refer to only A (optionally including elements other than B) in one embodiment; to only B (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment, etc.

[0108] As used herein in this specification and claims, "or" should be understood to have the same meaning as the meaning of "and / or" as defined above. For example, when the items in the list are separated, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one element in many elements or element lists, but also including more than one element and optionally other unlisted items. Only explicitly indicating the opposite term, such as "only one of..." or "exactly one of..." or when used in the claims, "consisting of..." will refer to including exactly one element in multiple elements or element lists. In general, when there are exclusive terms before, such as "any one", "one of...", "only one of..." or "exactly one of...", the term "or" used in this article should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, not two"). When used in the claims, "consisting essentially of..." should have the common meaning as used in the field of patent law.

[0109] As used in this specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to refer to at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one element of each element specifically listed in the list of elements and not excluding any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether or not related to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B" or equivalently "at least one of A and / or B") may refer to at least one (optionally including more than one) A in one embodiment, without B (and optionally including elements other than B); to at least one (optionally including more than one) B in another embodiment, without A (and optionally including elements other than A); to at least one (optionally including more than one) B in another embodiment, without A (and optionally including elements other than A); to at least one (optionally including more than one A) and at least one (optionally including more than one) B (and optionally including other elements) in yet another embodiment, etc.

[0110] In the claims and the above description, all transitional phrases, such as "includes," "comprising," "carrying," "having," "containing," "involving," "containing," "consisting of," etc., shall be understood as open-ended, i.e., including but not limited to. As provided in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

Claims

1. An integrated chromatography-spectroscopy system, comprising: a gas chromatograph for separating analytes into components, the gas chromatograph having a column in fluid communication with a carrier gas source that supplies a carrier gas to propel the components through the column; A molecular rotation resonance spectrometer, the molecular rotation resonance spectrometer being configured to measure a molecular rotation resonance spectrum of the component, the molecular rotation resonance spectrometer comprising: a measuring chamber for holding the component; a nozzle in fluid communication with the column and the measurement chamber to inject the component into the measurement chamber; and a microwave source in electromagnetic communication with the measurement chamber to excite the species under measurement with an excitation pulse having at least one spectral component in a frequency range of 6 GHz to 18 GHz; and a processor operably coupled to the molecular rotation resonance spectrometer to resolve the components of the analyte based on the molecular rotation resonance spectra of the components, wherein the processor is configured to resolve the components based on the molecular rotation resonance spectra of co-eluting components of the analyte.

2. The integrated chromatography-spectroscopy system according to claim 1, wherein the microwave source is configured to excite different components with excitation pulses of different frequencies.

3. The integrated chromatography-spectroscopy system according to claim 1, wherein the carrier gas source is a first carrier gas source, and the carrier gas is a first carrier gas, and the integrated chromatography-spectroscopy system further comprises: A second carrier source is in fluid communication with the nozzle to supply a second carrier gas to inject the component into the measurement chamber, the second carrier gas rotationally cooling the component.

4. An integrated chromatography-spectroscopy system comprising: a gas chromatograph for separating analytes into components, the gas chromatograph having a column in fluid communication with a carrier gas source that supplies a carrier gas to propel the components through the column; A molecular rotation resonance spectrometer, the molecular rotation resonance spectrometer being configured to measure a molecular rotation resonance spectrum of the component, the molecular rotation resonance spectrometer comprising: a measuring chamber for holding the component; a nozzle in fluid communication with the column and the measurement chamber to inject the component into the measurement chamber; and a microwave source in electromagnetic communication with the measurement chamber to excite the species under measurement with an excitation pulse having at least one spectral component in a frequency range of 6 GHz to 18 GHz; a processor operably coupled to the molecular rotation resonance spectrometer to resolve the components of the analyte based on the molecular rotation resonance spectra of the components, and A chiral tag source is connected to the nozzle fluid to inject a chiral tag into the measurement chamber together with the components, wherein the chiral tag binds to different enantiomers of at least one of the components, thereby generating a shift in the molecular rotation resonance spectrum of the different enantiomers that can be resolved by the molecular rotation resonance spectrometer.

5. An integrated chromatography-spectroscopy system comprising: a gas chromatograph for separating analytes into components, the gas chromatograph having a column in fluid communication with a carrier gas source that supplies a carrier gas to propel the components through the column; A molecular rotation resonance spectrometer, the molecular rotation resonance spectrometer being configured to measure a molecular rotation resonance spectrum of the component, the molecular rotation resonance spectrometer comprising: a measuring chamber for holding the component; a nozzle in fluid communication with the column and the measurement chamber to inject the component into the measurement chamber; and a microwave source in electromagnetic communication with the measurement chamber to excite the component under measurement with an excitation pulse having at least one spectral component in the frequency range of 6 GHz to 18 GHz; a processor operably coupled to the molecular rotation resonance spectrometer to resolve the component of the analyte based on the molecular rotation resonance spectrum of the component, an auxiliary detector operably coupled to the column and the processor to detect peaks in the output of the column, and Wherein the processor is configured to trigger measurement of the molecular rotation resonance spectrum in response to the peak and / or to process the molecular rotation resonance spectrum based at least in part on the peak.

6. An integrated chromatography-spectroscopy system comprising: a gas chromatograph for separating analytes into components, the gas chromatograph having a column in fluid communication with a carrier gas source that supplies a carrier gas to propel the components through the column; A molecular rotation resonance spectrometer, the molecular rotation resonance spectrometer being configured to measure a molecular rotation resonance spectrum of the component, the molecular rotation resonance spectrometer comprising: a measuring chamber for holding the component; a nozzle in fluid communication with the column and the measurement chamber to inject the component into the measurement chamber; and a microwave source in electromagnetic communication with the measurement chamber to excite the species under measurement with an excitation pulse having at least one spectral component in a frequency range of 6 GHz to 18 GHz; a processor operably coupled to the molecular rotation resonance spectrometer to resolve the components of the analyte based on the molecular rotation resonance spectra of the components, and A display is operably coupled to the processor to display a relationship of peaks in the column output to a plurality of molecular rotation resonance spectra.

7. An integrated chromatography-spectroscopy system comprising: a chromatograph for separating an analyte into components, wherein the components include co-eluting components that cannot be separated by the chromatograph; a molecular rotation resonance spectrometer in fluid communication with the chromatograph to measure a molecular rotation resonance spectrum of at least one of the components; as well as A processor is operably coupled to the molecular rotation resonance spectrometer to resolve co-eluting components from the chromatograph based at least in part on the molecular rotation resonance spectrum.

8. The system of claim 7, wherein the chromatograph is a gas chromatograph.

9. The system of claim 8, wherein the molecular rotation resonance spectrometer is a microwave molecular rotation resonance spectrometer having an expansion chamber, and the system further comprises: A carrier gas source is in fluid communication with the gas chromatograph and the resonant measurement cavity to push the analyte through the gas chromatograph and at least one of the components into the expansion chamber.

10. The system of claim 9, wherein the expansion chamber comprises a resonant cavity.

11. The system of claim 8, wherein the molecular rotation resonance spectrometer comprises a flow cell for receiving the components of the analyte from the gas chromatograph.

12. The system of claim 7, wherein the chromatograph is a liquid chromatograph.

13. The system of claim 12, wherein the molecular rotation resonance spectrometer is a microwave molecular rotation resonance spectrometer having an expansion chamber, and the system further comprises: a volatilization interface, the volatilization interface being in thermal communication with the liquid chromatograph to volatilize at least one of the components; as well as A carrier gas source is in fluid communication with the volatilization interface to push at least one of the components out of the volatilization interface and into the expansion chamber.

14. The system of claim 7, wherein the molecular rotation resonance spectrometer is configured to measure a spectrum having a bandwidth of at least 50 MHz of at least one eluate exiting the chromatograph.

15. The system of claim 7, further comprising: A processor is operably coupled to the molecular rotation resonance spectrometer to form a total molecular chromatogram of the analyte based at least in part on the molecular rotation resonance spectrum of the component.

16. The system of claim 7, further comprising: A processor operably coupled to the molecular rotation resonance spectrometer to resolve at least one of isomers, isotopologues, or isotopomers in the components of the analyte based at least in part on the molecular rotation resonance spectrum.

17. An integrated chromatography-spectroscopy system comprising: a chromatograph for separating an analyte into components, wherein the components include co-eluting components that cannot be separated by the chromatograph; a molecular rotation resonance spectrometer in fluid communication with the chromatograph to measure a molecular rotation resonance spectrum of at least one of the components; an auxiliary detector operably coupled to the chromatograph to detect peaks in an output of the chromatograph; as well as A processor is operably coupled to the molecular rotation resonance spectrometer and the detector to trigger measurement of the molecular rotation resonance spectrum in response to the peak and / or to process the molecular rotation resonance spectrum based at least in part on the peak.

18. The system of claim 7, further comprising: A nozzle is in fluid communication with the chromatograph and the molecular rotation resonance spectrometer to rotationally cool molecules of the at least one of the components before the molecular rotation resonance spectrometer measures the molecular rotation resonance spectrum.

19. A method for analyzing an analyte, the method comprising: separating the analyte into components using a chromatograph, wherein the components include co-eluting components that cannot be separated using the chromatograph; measuring a molecular rotation resonance spectrum of at least one of the components with a molecular rotation resonance spectrometer in fluid communication with the chromatograph; as well as At least one of isomers, isotopologues, or isotopomers in the components of the analyte are resolved based at least in part on the molecular rotation resonance spectrum.

20. The method of claim 19, wherein separating the analyte into components comprises sampling a continuous flow of the analyte with the chromatograph.

21. The method according to claim 20, further comprising: detecting peaks in an output of the chromatograph; as well as Measurement of the molecular rotational resonance spectrum is triggered in response to detecting the peak.

22. The method of claim 19, wherein measuring the molecular rotation resonance spectrum of at least one of the components comprises measuring a broadband spectrum of an eluate exiting the chromatograph.

23. The method of claim 19, further comprising: rotationally cooling the molecules of the at least one of the components prior to measuring the molecular rotational resonance spectrum of the at least one of the components, and Wherein measuring the molecular rotation resonance spectrum comprises exciting the at least one of the components with a microwave excitation signal.

24. The method of claim 19, further comprising: At least one unknown component of the analyte is identified based at least in part on the molecular rotation resonance spectrum.

25. The method of claim 19, further comprising: The at least one of the components is quantified based at least in part on the molecular rotation resonance spectrum.

26. The method of claim 19, further comprising: A total molecular chromatogram of the analyte is formed based at least in part on the molecular rotation resonance spectrum.

27. The method of claim 19, wherein the chromatograph is a gas chromatograph, the molecular rotation resonance spectrometer is a microwave molecular rotation resonance spectrometer having an expansion chamber, and the method further comprises: A carrier gas is used to propel the analyte through the gas chromatograph and to push at least one of the components into the expansion chamber.

28. The method of claim 19, wherein the chromatograph is a liquid chromatograph, the molecular rotation resonance spectrometer is a microwave molecular rotation resonance spectrometer having an expansion chamber, and the method further comprises: volatilizing at least one of the components; as well as The at least one of the components is pushed out of the volatilization interface and into the expansion chamber using a carrier gas.

29. A method of analyzing an analyte, the method comprising: separating the analyte into components using a chromatograph, wherein the components include co-eluting components that cannot be separated using the chromatograph; measuring a molecular rotation resonance spectrum of at least one of the components with a molecular rotation resonance spectrometer in fluid communication with the chromatograph; attaching a chiral tag to said at least one of said components prior to measuring said molecular rotational resonance spectrum; as well as Enantiomers in the component of the analyte are identified based on the molecular rotation resonance spectrum and / or enantiomeric excess of the component of the analyte is determined based on the molecular rotation resonance spectrum.

30. An integrated chromatography-spectroscopy system comprising: a gas chromatograph for separating an analyte into components; a molecular rotation resonance spectrometer in fluid communication with the gas chromatograph to measure a molecular rotation resonance spectrum of the component; as well as a pulse jet ultrasonic expansion source in fluid communication with a column of the gas chromatograph and a vacuum chamber of the molecular rotational resonance spectrometer to transfer the component from the column to the vacuum chamber, The molecular rotation resonance spectrometer is configured to resolve isomeric compounds by molecular rotation resonance spectroscopy analysis.

31. The system of claim 30, wherein the pulsed jet ultrasonic expansion source is configured to rotationally cool molecules of the analyte for molecular rotation resonance analysis by the molecular rotation resonance spectrometer.

32. The system of claim 30, wherein the pulsed jet ultrasonic expansion source is configured to rotationally cool the molecules of the analyte to a temperature below 10 K for molecular rotation resonance spectroscopy analysis by the molecular rotation resonance spectrometer.

33. The system of claim 30, wherein the molecular rotation resonance spectrometer is configured to analyze analytes having a molecular weight greater than 150 amu.

34. The system of claim 30, wherein the molecular rotation resonance spectrometer is configured to resolve components that co-elute from the gas chromatograph.

35. An integrated chromatography-spectroscopy system comprising: a gas chromatograph for separating an analyte into components; a molecular rotation resonance spectrometer in fluid communication with the gas chromatograph to measure a molecular rotation resonance spectrum of the component; as well as a pulse jet ultrasonic expansion source in fluid communication with a column of the gas chromatograph and a vacuum chamber of the molecular rotational resonance spectrometer to transfer the component from the column to the vacuum chamber, Wherein the system is configured to identify and quantify the component in the absence of a reference standard.

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