Highly selective chromatography-molecular rotational resonance spectroscopy system and method

Abstract

The capabilities of gas or liquid chromatography-molecular rotational resonance (GC / LC-MRR) instruments exceed those of high-resolution mass spectrometry and nuclear magnetic resonance instruments in terms of selectivity, resolution, and compound identification. MRR detection offers high selectivity for selective gas- or liquid-phase separations, including the ability to resolve coeluting peaks and isomeric compounds without compromising specificity or precision. MRR can perform both qualitative identification and absolute quantification of analyte components separated by GC or LC without reference standards. GC-MRR is ideal for compound-specific isotope analysis (CSIA), allowing the identification of enantiomers and enantiomeric excess. GC-MRR measurements are particularly useful for studying biosynthesis / degradation and geochemical isotope identification of compounds.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. application Ser. No. 62 / 977,846, entitled "Molecular Rotational Resonance Spectrometer for Measurement of Gas Chromatography Effluents," filed Feb. 18, 2020; Ser. No. 62 / 913,082, entitled "A Molecular Detection / Separations System of Singular Specificity," filed Oct. 9, 2019; and Ser. No. 62 / 844,280, entitled "Hyphenated MRR Spectroscopy: MRR Spectroscopy as a Chromatographic Detector," filed May 7, 2019. Each of these applications is incorporated herein by reference in its respective entirety. [Background technology]

[0002] Chromatographic separation techniques are commonly used to determine and quantify components in chemical mixtures. 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 in the drug development and approval process and to measure impurities in pharmaceutical products. The Food and Drug Administration (FDA) relies heavily on gas chromatography for regulatory validation.

[0003] Conventional chromatography devices include columns, which contain stationary phases that retain different analytes in the column for different lengths of time based on their weight, polarity, or other properties. This allows these components to be separated. Conventional chromatography devices also include detectors that return signals as the chemical components are eluted from the column. Some of these detectors do not obtain chemical information, such as flame ionization detectors (FIDs), which return signals by burning the analytes. Others, such as mass spectrometry (MS), obtain chemical-specific information. However, for all detectors, the column must completely or nearly completely separate the individual components of a mixture in order to reliably quantify the components. Most detectors cannot accurately resolve and quantify the individual components when multiple components elute from the column simultaneously. Thus, great efforts are being made to develop new columns that can better separate messy mixtures, and to develop methods that allow analytical chemists to completely separate the individual components. Despite these enormous efforts, there are certain important analyses for which chromatographic separation is impossible or very difficult (requiring expensive columns and long run times).

[0004] Molecular rotational resonance (MRR) spectroscopy, also known as molecular rotational spectroscopy or microwave spectroscopy, characterizes compounds by their pure rotational angular momentum transfer in the gas phase. The rotational energy levels of a molecule are quantized as defined by its three-dimensional mass distribution and expressed as its moment of inertia, I. This can be expressed as (in one dimension) I=Σm i r i 2 where m is defined as 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 in the three spatial axes, and therefore, using rotational spectroscopy, molecules can be clearly distinguished by their structural differences. Their many very narrow spectral lines (typically, the resolution of the spectrum is ν / Δv ≈ 10 -5), high-resolution rotational spectra are therefore completely unique for each molecular structure.

[0005] For example, Figure 1 shows the isotopomers of the same mass, each of which has a clearly resolved spectral pattern. 13 CH 3 CN and CH 3 13 The MRR spectrum of CN is shown. Notably, the MRR spectrum of each compound can be calculated with high accuracy. In this way, it is possible to use MRR to unambiguously identify compounds without a reference sample. Summary of the Invention

[0006] 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 systems, also referred to as GC-MRR or LC-MRR instruments, can measure spectra orders of magnitude faster than other MRR or rotational spectroscopy systems using broadband MRR measurement techniques, including chirped-pulse FT (CP-FT) techniques. The GC-MRR or LC-MRR instruments of the present invention have at least three advantages over other GC or LC detection systems, particularly mass spectrometry (MS): (i) MRR is highly sensitive to differences in molecular structure and therefore can resolve all types of isomeric compounds; (ii) MRR can resolve and quantify co-eluting compounds without loss of specificity or precision; and (iii) both qualitative identification and absolute quantification can be achieved without reference standards.

[0007] The instrument of the present invention may take the form of an integrated chromatography-spectroscopy system comprising a gas chromatograph, an MRR spectrometer, and a processor operatively coupled to the MRR spectrometer. In operation, the gas chromatograph separates an analyte into components in a column. The column is in fluid communication with a carrier gas source that provides a carrier gas to pass the components through the column. The MRR measures a chromatogram of the components. It comprises a measurement chamber, a nozzle, and a microwave source. The measurement chamber holds the components of the analyte separated by the gas chromatograph. A nozzle in fluid communication with the column and the measurement chamber injects the components into the measurement chamber. And, a microwave source in electromagnetic communication with the measurement chamber excites the components in the measurement with an excitation pulse having at least one spectral component in a frequency range of about 6 GHz to about 18 GHz. The processor analyzes the components of the analyte based on the MRR spectrum of the components.

[0008] Another instrument includes a chromatograph and an MRR spectrometer in fluid communication with the chromatograph, the chromatograph separates an analyte into components, and the MRR spectrometer measures an MRR spectrum of at least one of the components.

[0009] Another instrument of the invention includes a gas chromatograph, an MRR spectrometer, and a pulse jet expansion source in fluid communication with the column of the gas chromatograph and the vacuum chamber of the MRR spectrometer. Again, the gas chromatograph separates the 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 to the vacuum chamber.

[0010] In the instrument of the present invention, the raw data collected by the MRR spectrometer is a series of successive time-domain free induction decay (FID) traces from the chromatographically separated analyte components. Each of these time-domain FID traces can be Fourier transformed to obtain a corresponding molecular rotational resonance (MRR) spectrum using a processor (e.g., the instrument processor or another processor). The processor identifies the lines of each of these MRR spectra and sums the amplitudes of the lines of each MRR spectrum to obtain amplitude values ​​corresponding to the time bins of the time-domain FID trace. This forms a chromatogram of the analyte components based on the amplitude values ​​and time bins of the time-domain FID trace. It also identifies peaks in the chromatogram and sums, integrates, or averages the time-domain FID traces corresponding to the time bins associated with the chromatographic peaks to obtain an integrated time-domain FID trace. The integrated time-domain FID traces are Fourier transformed to obtain the MRR spectrum associated with the chromatographic peaks.

[0011] All combinations of the above concepts and further concepts discussed in more detail below (provided such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter found at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein. It should also be understood that the terminology explicitly used herein and also found in any documents incorporated herein by reference should have the meaning most consistent with the specific concepts disclosed herein.

[0012] Those skilled in the art 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 to scale. In some cases, various aspects of the inventive subject matter disclosed herein may be shown 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). [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the molecular rotational resonance (MRR) spectra of two isotopomers of acetonitrile, showing selectivity for isobaric compounds as well as very high resolution of the MRR spectral lines. [Diagram 2] Figure 2A shows chlorinated dioxin isomers that have not been separated by gas chromatography (GC) but can be analyzed by GC-MRR spectroscopy. Figure 2B shows an approved fluorinated drug and its possible de-F degradation products that can be analyzed by GC-MRR spectroscopy. Figure 2C shows a chromatogram of co-eluting acetonitrile isotopic species that can be analyzed by GC-MRR spectroscopy. [Figure 3A] FIG. 3A is a schematic diagram of the GC-MRR system. [Figure 3B] FIG. 3B is a photograph of an MRR spectrometer suitable for use in the GC-MRR system of FIG. 3A. [Figure 3C] 3C and 3D show diagrams of graphical user interfaces suitable for displaying MRR spectra obtained using the GC-MRR system of FIG. 3A and / or the MRR spectrometer of FIG. 3B. [Figure 3D] Same as above. [Figure 4] FIG. 4 shows an example of a pulse jet nozzle suitable for use in a GC-MRR system. [Diagram 5] Figure 5A shows the simulated MRR spectra of isopulegol at four different temperatures, showing an increase in sensitivity and a shift to lower frequencies as the rotational temperature decreases. Figure 5B shows a close-up of the simulated MRR spectrum of Figure 5A over the frequency range 6-18 GHz. At room temperature, this molecule is completely undetectable at microwave frequencies, as the line intensities are below the detection cutoff of 10-7 units. [Figure 6] FIG. 6 shows simulated MRR spectra of molecules with different molecular weights at a temperature of 300K. [Figure 7A] FIG. 7A shows the process for calculating total molecular chromatograms (TMC) and extracted molecular chromatograms (EMC) from raw time-domain free induction decay (FID) traces collected by the integrated chromatography-MRR system. [Figure 7B] FIG. 7B is a schematic diagram of the TMC and EMC calculated from the FID trace. [Figure 7C] FIG. 7C shows the TMCs of 24 isotopologues of five common organic molecules obtained using the GC-MRR system. [Figure 7D] FIG. 7D shows the EMCs of the 24 isotopologues in the TMC of FIG. 7A. [Figure 8] Figure 8A shows the TMCs of (a) bromoethane, (b) thiophene, (c) 2-chlorothiophene, (d) 3-chlorothiophene, (e) 3-chloropyridine, and (f) 2-chloropyridine. Figure 8B shows two examples of how the natural isotopic abundances of the compounds in Figure 8A can be easily obtained by GC-MRR EMC. The error of these ratios is ±3% RSD. [Figure 9] FIG. 9 shows a hybrid liquid chromatography (LC)-MRR spectroscopy system. [Figure 10] Figure 10A shows chiral tagging, which is the gas-phase formation of different diastereomers with different spectra by combining chiral tags with enantiomers. Figure 10B shows the enantiomeric excess analysis from MRR spectroscopy. Measurements were performed using racemic or even pure enantiomers of the chiral tags. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [Analytical problems that can be solved by GC-MRR] While GC, LC, and MRR spectroscopy are well-established techniques, MRR spectroscopy is still in its infancy in terms of commercial use for analytical applications. Nevertheless, GC-MRR and LC-MRR spectroscopy can solve problems that other techniques or combinations of techniques cannot currently address. Direct MRR spectroscopy, without chromatographic separation, has been shown to identify components in mixtures containing up to about 10 components. However, real samples in biochemistry, environmental, petrochemistry, and other application areas where GC and LC are used have matrices with many analytes at various concentrations. Furthermore, MRR spectra of mixtures are highly complex, in part because the patterns of each component interleave with each other. Identification of unknown components in a mixture is typically done by computer-assisted pattern recognition, which is very time-consuming when many species are present, ultimately limiting the analyst's ability to interpret complex data. Thus, GC-MRR or LC-MRR hyphenation greatly expands the complexity of samples that can be successfully analyzed by MRR spectroscopy and allows accurate quantification of mixture components using the areas of chromatographic peaks in the MRR chromatogram.

[0015] At the same time, when used as a detector, MRR spectroscopy brings state-of-the-art capabilities to gas and liquid chromatography analysis, and in particular, MRR spectroscopy has particular advantages over other GC and LC detectors when chromatographic separation is impossible or difficult, since MRR spectroscopy can easily identify and quantify individual components that cannot be separated chromatographically.

[0016] The challenge of peak co-elution or partial overlap is fundamental in separation science. Gidding used statistical overlap theory to make the grim prediction that if a chromatographic column with a peak capacity of n is used, separation of n compounds is "practically hopeless" due to peak overlap. This theoretical challenge is supported by the general resolution problem of chromatography: no single column can separate all the components in a complex mixture under given conditions. This statement is especially true for today's much more complex separations that include isomeric components. Although incredible advances have 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 co-eluting compounds is a significant advance for analytical chemistry.

[0017] Advanced mathematical approaches exist for separating overlapping peaks with area extraction, such as Fourier self-deconvolution, wavelets, multivariate curve analysis, and iterative curve fitting. However, most of these chemometric techniques do not work when (a) the peaks completely overlap, or (b) no experimentally measured reference spectrum of each component exists. This is where a highly selective detector such as MRR spectroscopy is very useful: MRR spectroscopy does not rely on chemometrics to resolve coelution. Instead, it produces a signal that is so specific for the analyte that no other molecule will match it. Furthermore, MRR spectroscopy is selective enough that it can distinguish between two molecules with the same mass, e.g., isotopomers, diastereomers, and enantiomers. This is something that even the best mass spectrometry detectors for chromatography cannot do.

[0018] Figures 2A-2C show a few examples where GC-MRR and LC-MRR can address difficult challenges in separation science that cannot be solved by GC or LC alone. Figure 2A shows two isomers of tetrachlorodibenzodioxin (TCDD), a persistent organic pollutant and human carcinogen. To date, there is no GC column capable of separating 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 generate distinct spectra for these compounds.

[0019] As a second example, fluorine is becoming increasingly important in pharmaceuticals, and fluorinated compounds can be very difficult to analyze by GC from their defluorinated counterparts. However, it is easy to distinguish these compounds by rotational spectroscopy. Figure 2B shows the structure of bictegravir, a recently FDA-approved drug, for which GC-MRR can be highly advantageous to analyze fluorinated / defluorinated impurities.

[0020] As another example, FIG. 2C shows the concentration of acetonitrile (CH) from a GC-MRR spectroscopy display. 3 The figure shows a chromatogram of a mixture of eight isotopologues of fully deuterated acetonitrile (CDCN). Compound-specific isotope ratio analysis is important in many research areas, including metabolism and environmental degradation studies. Chromatographically, fully deuterated acetonitrile (CDCN) is the most widely used isotope ratio for the determination of the isotope ratio of the acetonitrile-containing acetonitrile. 3 Only one isotopologue (CN) can be separated from a mixture of isotopologues. Mass spectrometry can be used to distinguish between isotopologues with different masses, but even the most powerful mass spectrometers cannot separate the two. 13 C-substituted isotopomers, 13 CH 3 CN vs CH 3 13 CN cannot be distinguished.

[0021] GC-MRR metabolomics studies (described in more detail below) have shown that different bacterial cultures in groundwater can have very distinct isotopic selectivities for the same organic compounds. Furthermore, the chemometric methods mentioned above, including the effective multivariate curve analysis methods, do not work in this case due to the high degree of overlap. This study is an example of the investigation of biosynthesis and degradation that can be performed with GC-MRR spectroscopy but not by other methods.

[0022] [GC-MRR Spectroscopic Measurement Type and Bands] GC-MRR spectroscopy systems can be configured to perform targeted measurements, broadband measurements (e.g., measurements over several spectral lines and / or bandwidths of 50 MHz, 100 MHz, or more), or both. Targeted measurements are similar to selected ion measurements in mass spectrometry and typically focus on capturing the MRR spectrum of a particular species or set of species with good sensitivity. Broadband measurements are similar to total ion monitoring measurements in mass spectrometry and are often 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 from the sample in response to the chirped pulse. 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: 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." Broadband measurements can be performed across the entire band at once or across segments of the band (e.g., 2 GHz or 4 GHz segments of the 6-18 GHz band) to reduce data rates, as 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.

[0023] Targeted MRR measurements are typically performed when the goal is to determine whether a sample contains a particular compound or contains a compound. Instead of irradiating the sample with chirped pulses, the MRR instrument irradiates the sample with one or more narrowband (e.g., single frequency) pulses and detects the FID signal(s) that emanate from the sample in response to these pulses. Because the measurements are targeted, the instrument can acquire, process, and store data more quickly than broadband measurements, for example, at 2 Hz, 3 Hz, 5, Hz, 10 Hz, or more. This measurement rate can be increased by performing a Fourier transform directly on the field programmable gate array (FPGA) that acquires the data.

[0024] Since the excitation frequency and target species are known in advance, targeted MRR instruments can operate without a "species recognition" function. Targeted MRR measurements typically involve examining the intensity of a single line versus time, since the measured intensity is proportional to the concentration of the species. If the target species and GC / LC separation parameters are known in advance, the MRR spectrometer can be preprogrammed to target different spectral lines or bands of different species. Consider quantification of a mixture of compounds A and B. If compound A exits the column 10 seconds after the start of the measurement and has a strong resonance at 10 GHz, the MRR spectrometer may be programmed to interrogate the sample at 10 GHz with a measurement time of 10 seconds. Similarly, if compound B exits the column 15 seconds after the start of the measurement and has a strong resonance at 6.8 GHz, the MRR spectrometer may be programmed to interrogate the sample at 6.8 GHz with a measurement time of 15 seconds. If there is an auxiliary detector at the column output, the auxiliary detector can trigger targeted measurements based on peak / eluent order (e.g., first eluent measured at 7 GHz, second eluent measured 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).

[0025] Targeted and broadband measurements can be performed in a variety of frequency ranges, including bands in the microwave range (e.g., over the 6-18 GHz band) or the millimeter wave range (e.g., 75-110 GHz, 260-290 GHz, or 520-580 GHz). The measurement type and measurement bands vary depending on the analyte and the information desired.

[0026] 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 typically performed using a flow cell as the measurement chamber, which limits the molecular weight of the analyte components to about 120 amu. In addition, it usually takes several seconds for the gas to travel through the flow cell, reducing the time resolution of the GC output, which typically elutes components in less than a second. The GC carrier gas also dilutes the sample in flow cell measurements, reducing sensitivity.

[0027] As described below, microwave MRR measurements can be performed using an expansion chamber fed by an ultrasonic expansion nozzle. This allows for faster measurements and measurement of higher molecular weight molecules while preserving the time resolution of the GC output, as described below. In addition, 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 extra dilution of the sample or reduce the measurement sensitivity.

[0028] [GC-MRR Spectroscopic System] 3A and 3B show a hyphenated GC-MRR instrument 300 capable of analyzing messy and / or complex mixtures in a single run. Compound-specific isotope analysis (CSIA) can be performed, including isotope fractionation and quantification that cannot be performed by other methods. For example, isomers and other compounds that cannot be separated using GC alone can be identified without references.

[0029] The GC-MRR instrument 300 includes a gas chromatograph 310 coupled to an MRR spectrometer 320 with 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 constituents including isomers, isotopes, isotopomers, and isotopologues, through the column 314 via the flow interface 330 and into the MRR spectrometer 320. The analytes may be (periodically) siphoned off a continuous stream or flow of gas or liquid, accumulated and volatilized as appropriate, and then injected into the column 314 such that the gas chromatograph effectively samples a continuous stream in the same way that an analog-to-digital converter (ADC) samples an analog signal.

[0030] Some or all of the components of the analyte may propagate through the column 314 at different rates and therefore appear at the end of the column 314 at different times. If these times are widely separated enough, the components can be analyzed at the output of the column 314. Other components, for example isomers, may co-elute, i.e., they may appear at the same or near the same time at the output of the column 314, and therefore cannot be analyzed using GC alone.

[0031] The carrier gas pushes the (at least partially separated) components into the measurement chamber 324 of the MRR spectrometer via an interface 330 so that the MRR spectrometer 320 can measure the MRR spectrum of the components. This interface 330 allows the sample to be injected into the MRR spectrometer 320 either via a GC column 314 or directly (e.g., for pure compounds or simple mixtures where GC separation is not required). In other words, some samples may require GC separation, while others may not. Samples that do not require GC separation can be injected directly into the sample MRR spectrometer (without going through a GC), while other samples can be injected through a GC.

[0032] For example, the measurement chamber 324 may be a flow cell with at least a pair of holes that allow the gas phase components and carrier gas to enter and exit. Alternatively, a pulse jet expansion nozzle, a continuous wave jet, or a buffer gas cooling cell may introduce the components into the measurement chamber 324 while simultaneously spin-cooling the components for better measurement performance, as described in more detail below. The MRR spectrometer 320 measures the MRR spectrum of each component by subjecting it to one or more excitation pulses of microwave and / or millimeter wave radiation. The excitation pulses are generated by a signal generator 321, such as an arbitrary waveform generator, a direct digital synthesizer, or a pulse pattern generator, and may be filtered, frequency multiplied, and / or upconverted using optional circuitry 322. A source 323 applies the excitation pulses to the molecules in the measurement chamber 324.

[0033] The interface 330 may also be coupled (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 to push or propel the analyte components into the measurement chamber 324 of the MRR spectrometer. The first and second carrier gases 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.

[0034] The interface 330 can also be coupled (in fluid communication) with a (chiral) tag source 354. By mixing the chiral tags from the chiral tag source 354 with the analyte components in the reservoir at the interface 330, the chiral tags attach themselves to the different components. The chiral tags alter the moment of inertia of the different enantiomers between the analyte components, allowing the enantiomers to be resolved and quantified from their MRR spectra, as described below. The tag source 354 can store and supply other types of tags, including polar molecules for tagging apolar molecules that do not have an MRR spectrum, to produce complexes that have a dipole moment and can therefore be detected by MRR.

[0035] The excited molecules emit coherent radiation in response to excitation at their characteristic rotational frequency by free induction decay (FID) in a few microseconds. The receiver 326 detects the analog FID signal, which is digitized by the ADC 327. The processor 340 (e.g., a field programmable gate array (FPGA)) records the digitized FID signal in the time domain and Fourier transforms it to the frequency domain to generate the MRR spectrum of the components. Using a library of MRR spectra, the 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.

[0036] Current MRR instruments measure volatile liquids whose sample composition does not change significantly with time. The pulse jet source of microwave MRR instruments typically operates at a repetition rate of 10 Hz, but the instruments record spectra by signal averaging single lines for seconds to minutes. On the other hand, for GC-MRR spectroscopy, the peak shapes are much narrower (a few seconds or even less than a second), and therefore the instrument 300 records data with good time resolution (e.g., at a sampling rate of 5-10 Hz) thanks to a digitizer (ADC 327) that can achieve the desired throughput rate and a processor 340 that can perform data handling operations without dead time.

[0037] Processor 340 may continuously measure and record the MRR spectrum of the effluent as it exits GC column 314. In some cases, processor 340 records and processes all of the MRR spectrum. In other cases, processor 340 records all of the time domain data and Fourier transforms only those segments that correspond to "interesting" output from GC column 314 to conserve processing resources and reduce total processing time. Processor 340 may discard unprocessed or unexamined time domain and / or Fourier domain data.

[0038] Alternatively, an auxiliary (universal) detector 350, such as a thermal conductivity detector (TCD), can trigger the MRR measurement based on the output of the GC column 314. This auxiliary detector 350 can be either in-line (sampling the same gas stream) as shown in FIG. 3A, or split (such as an FID or MS detector where the analyte is destroyed). When the auxiliary detector 350 detects a peak in the GC output, it sends a trigger signal to the processor 340, which then triggers the emission of an excitation pulse(s) by the signal generator 321 and measures and analyzes the resulting FID signal. The processor 340 can also record all of the MRR data, as described above, and discard MRR data that does not map 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 a component without a dipole moment, while isotopic information from the MRR spectrometer 320 can complete the characterization of the other components.

[0039] For details on triggering MRR spectroscopy measurements, see, for example, U.S. Pat. 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.

[0040] FIG. 3B is a photograph of a sample instantiation of the measurement chamber 324 and MRR spectrometer 320 in a GC-MRR spectroscopy system 320. This MRR spectrometer 320 is depicted as a microwave MRR spectrometer configured to perform targeted measurements. In this image, the sample inlet 332 is on the left side of the measurement chamber 324. Two spherical mirrors 328 are at the left and right ports, and an ultrasonic expansion nozzle 334 is mounted in one port, while the other is held above vacuum on an automated translation stage 344 controlled by a processor 340. The mirror 328 makes the measurement cavity 324 a resonant Fabry-Perot cavity. The translation stage 344 adjusts 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 mirror 328. These horns can be mounted at right angles to the target mirror 328. One horn can be attached to the flange 329 closest to the reader in Figure 6, and the other can be attached to the opposite side (not shown). Measurement parameters, including translation stage and excitation frequency settings, as well as measurement results, can be displayed by a display 342 operably coupled to processor 340 (Figure 3A).

[0041] 3C and 3D show two views of a user interface that can be displayed on the display 342. The view shown in FIG. 3C shows the flow of a user interface for acetonitrile (CH 3 The interface shows the broadband millimeter wave MRR spectrum of a mixture of eight isotopologues of 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,19,20,21,22,19,23,24,25,26,27,28,30,29,31,29,32,29,33,34,35,40,41,42,14,29,35,42,15,36,37,43,16,38,44,17,45,51,18,52,19,60,25,26,27,34,19,70,29,80,35,41,18,19,25,35,42,19,36,27,43,19,25,44,17,45,51,18,52,19,60,25,34,19,70,35,42,19, 3 Only one of the seven species (CN) can be separated from the others using GC; the remaining seven species run off the GC column at the same time (i.e., the other seven species are co-eluted) and cannot be analyzed using GC alone.

[0042] Because the eight acetonitrile isotopologues have distinct MRR spectra, the MRR spectrometer can analyze them even when they flow off the column simultaneously. The extracted MRR chromatograms of each isotopologue can be plotted and the peaks integrated to provide the exact concentration of each of the species. In this example, the eight isotopologues were mixed in equal concentrations and 5 nL of this mixture was injected directly into the MRR spectrometer for analysis. The MRR spectrometer measured and stored a broadband MRR spectrum of the mixture in less than a second. The information recorded in the MRR spectrum includes both the identity of all the isotopologues in the mixture and the amount of each in the gas cell.

[0043] [Molecular weight and rotational cooling for GC / MRR spectrometer connection] One of the challenges of GC-MRR spectroscopy systems is the upper molecular weight limit of the analytes. For GC-MRR spectroscopy systems with flow cells operating at room temperature or above, the upper molecular weight limit may be around 150 amu, and sensitivity at molecular weights between 100 and 150 amu may be limited, since MRR spectra of molecules at room temperature tend to be very weak above 150 amu. However, by using a pulsed jet ultrasonic expansion source, a continuous wave jet, or a buffer gas cooling cell, molecules are rotationally cooled for MRR analysis while keeping them in the gas phase. GC-MRR instruments with a pulsed jet expansion source, a continuous wave jet, or a buffer gas cooling cell can analyze molecules with higher molecular weights (e.g., up to 400 amu or more) thanks to this rotational cooling.

[0044] FIG. 4 shows a close-up view of a pulse-jet ultrasonic nozzle 400 suitable for injecting analytes into the measurement chamber of the MRR spectrometer of a GC-MRR system such as those shown in FIGS. 3A and 3B. There are three gas streams entering the ultrasonic expansion nozzle: a GC inlet 410 for analyte components from the GC column; a vent valve inlet 430 for rapid removal of solvent or other volatile matrix components; and an optional inlet 430 for (additional) carrier gas. The GC inlet 410 and vent valve inlet 420 can be made of 1 / 16″ tubing and can be heated to at least 300° C. to allow direct introduction of the GC column at that temperature. In FIG. 4, the tubing is PEEK plastic, but the connections can alternatively be made of metal(s) with better thermal properties. The central (1 / 4″) gas connection 430 for carrier gas is optional and can be used for purge gas to quickly clean the sample. All three gas streams are combined in a reservoir 440, which can have a volume of about 500 μL or less. By reducing the reservoir volume, extra-column spread can be reduced.

[0045] The combined gas stream is co-expanded with the rare carrier gas through a pinhole nozzle 450 with a diameter of about 1 mm. The pinhole nozzle 450 is rapidly opened and closed by a solenoid valve 452 sealed with a Teflon poppet. For each gas pulse (about 1 ms duration), the analyte component(s) is forced through the pinhole 450 into a high vacuum (about 10 -6 The molecules then travel to the MRR spectrometer measurement chamber, which is maintained at a constant pressure (T) of 1000 MPa (320 Torr). On their way through the pinhole 450, the molecules collide with the carrier gas multiple times. These collisions reduce the rotational temperature of the analyte components to about 1-2 K.

[0046] For example, when injecting analyte components separated by a GC column into a microwave MRR measurement chamber equipped with a pulse-jet ultrasonic nozzle, the carrier gas (e.g., neon) pressure can be set to about +2 to about +5 pounds per square inch gauge pressure (psig). The nozzle pulses at 10 Hz. During 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 a typical flow rate of a wide-bore GC column. Injecting neon carrier gas at the nozzle (e.g., via optional carrier gas inlet 430 in FIG. 4) can enhance pulsed valve operation. When neon carrier gas is injected into the nozzle, the GC column can be operated with different carrier gases, such as hydrogen or helium, since neon dominates the rotational cooling caused by ultrasonic pulse injection of the analyte components into the microwave MRR measurement chamber. Both carrier gases can be injected into the MRR measurement chamber.

[0047] Figures 5A and 5B show simulated MRR spectra of isopulegol (monoterpene and menthol intermediate, mass 154 amu) at four molecular temperatures: room temperature (300 K; bottom), 100 K (middle bottom), 20 K (middle top), and 2 K (top). Figure 5A shows the spectrum spanning microwave and millimeter wave frequencies from 0 to 600 GHz, and Figure 5B shows the spectrum in the microwave band from 0 to 18 GHz. These spectra show improved measurement performance at low temperatures, which can be achieved by using a pulse-jet ultrasonic expansion source to inject the GC output into the vacuum chamber of the MRR spectrometer of the GC-MRR spectroscopy system. Pulse-jet ultrasonic expansion with an ideal carrier gas (e.g., neon, which has excellent cooling properties) can achieve rotational temperatures of 1 K to 2 K (one tradeoff in using ultrasonic expansion sources is that they have a larger vacuum chamber to enable the expansion; however, the instrument size is comparable to high-end mass spectrometers).

[0048] The MRR spectra in Figures 5A and 5B show several effects that occur by cooling molecular rotations. First, the MRR spectrum is dramatically simplified. For every molecule, there are far fewer rotational energy levels and therefore far fewer transitions. This also means that more components can be resolved in a single mixture because there are fewer lines per molecule. Second, the MRR spectrum becomes orders of magnitude more intense (as can be seen from the y-axis in Figures 5A and 5B), which may lead to more sensitive and / or higher signal-to-noise ratio (SNR) measurements. Without being bound to a particular theory, the increase in intensity shown in Figures 5A and 5B is due to a smaller number of rotational vibrational states. Third, the MRR spectrum is shifted to lower frequencies. The shift in the spectrum to lower frequencies is also advantageous, since it is possible to achieve higher excitation power levels using less expensive components in the microwave region (especially below 18 GHz, e.g., 6-18 GHz) than in the millimeter wave region (e.g., 75 GHz and above). Nearly all molecules heavier than 50 amu have a spectral intensity peak in the microwave range (6-18 GHz).

[0049] Figure 6 shows simulated MRR spectra at room temperature for three different sized molecules. Generally, as the molecular size increases, the MRR spectrum becomes more complex due to the increased number of rotational and vibrational states. In the pulse jet, the vibrational states are also cooled, so the spectra of the larger molecules become less complex as shown above. It becomes easier to measure MRR spectra for compounds up to 300 amu, and even more is feasible (the main limitation becomes getting the compound in and keeping it in the gas phase).

[0050] [Total molecular chromatogram (TMC) and extended molecular chromatogram (TMC)] 7A shows a method 700 for processing raw data measured by the integrated GC-MRR instrument 300 shown in FIG. 3A (this method 700 can also be used to analyze LC-MRR data). As described above, the GC-MRR instrument 300 acquires time-domain data by injecting (at least partially) separated components from the GC column 314 into the measurement chamber 324 of the MRR spectrometer, then exciting the component(s) in the measurement chamber 324 with a microwave or millimeter wave pulse and recording the resulting time-domain free induction decay (FID) signal(s) (702). Over time, the instrument 300 records a series of these time-domain FID traces, for example, at 5 Hz or 10 Hz.

[0051] Each of these raw time-domain FID traces is transformed to obtain a set of MRR spectra (704), each of which is associated with a different time bin during the measurement period. The amplitudes of the spectral lines (peaks) of each of these MRR spectra are identified (706) and summed (708) to obtain an amplitude value for the time bin associated with that MRR spectrum. The amplitude values ​​are plotted as a function of time to obtain a total molecular chromatogram (TMC) (710). Like other chromatograms, the TMC represents the separation of components emerging from a chromatographic column as a function of time, with each peak corresponding to one or more components eluting from the column in that time bin.

[0052] Each peak in the TMC may span several time bins. Identifying the peaks and associated time bins (712) allows for the identification of the corresponding raw time domain FID traces, which can be summed or averaged (714) and then Fourier transformed to obtain the MRR spectrum associated with the peak (716). This MRR spectrum is called the extracted molecular chromatogram (EMC) and can be used to identify, analyze, and / or quantify the analyte components (718). For example, if the components are isomers that cannot be analyzed from the TMC, they can be analyzed by the differences in their MRR spectra in the EMC. Components can be identified by comparing the EMC to a library of previously measured EMCs (MRR spectra) and / or theoretical EMCs.

[0053] FIG. 7B shows the relationship between the FID data, the TMC, and the EMC. The TMC is plotted as a time series, with each point (amplitude value) corresponding 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 each time bin. In some cases, multiple analyte components may contribute to a given peak. In FIG. 7B, for example, components 1 and 2 both contribute to the left peak of the TMC and cannot be resolved from the peak itself. Component 1 contributes to the amplitude values ​​for time bins 1-8, and component 2 contributes to the amplitude values ​​for time bins 6-12. Components 1 and 2 can be resolved, identified, and quantified from the EMC of the left peak. This EMC is calculated by integrating or averaging the FID trace for time bins 1-12 and then Fourier transforming the integrated or averaged FID trace.

[0054] Figures 7C and 7D show a single GC-MRR analysis of 24 isotopic species of five common organic molecules, demonstrating the feasibility and utility of a GC-MRR instrument obtained using the process of Figure 7A. Figure 7C shows the TMC where the peak intensities of the observed transitions are summed. The TMC shows five main peaks and two small shoulders just before the main acetone and acetonitrile peaks (3.2 min and 7.6 min, respectively). These small partially resolved shoulders are fully deuterated acetone (acetone-d6) and tritiated acetonitrile (ACN-d-3), which are chromatographically separated from their related isotopologues and isotopomers.

[0055] Each point in the TMC contains a broadband MRR spectrum, allowing the individual contributions of the various species to be resolved. The extraordinary selectivity of MRR spectroscopy allows the TMC to be resolved into 24 EMCs, one EMC for each isotopically distinct compound, as shown in Figure 7D. In each EMC trace, the transition intensities at the frequency of that isotopic species only are summed. Since isotopes can be directly determined based on their moments of inertia, isotopic pattern matching / analysis is not necessary for molecular identification. 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 spectra due to their different moments of inertia.

[0056] FIG. 8A shows the TMCs obtained by GC-MRR spectroscopy and the process of FIG. 7A for bromoethane and five heterocyclic compounds. FIG. 8B shows the isotopologue-specific EMCs showing the natural isotopic abundances of bromoethane and 2-chloropyridine. Integration of the GC-MRR peak areas gives quantitative information for all species. The changes in dipole moments and rotational constants are typically small, although not used in the TMCs of FIG. 8A or the EMCs of FIG. 8B. The ratio between the MRR peak intensities can also be used directly as an accurate determination of the isotope ratios.

[0057] Unlike mass spectrometry, isobaric compounds do not cause problems. Furthermore, peak (compound) coelution does not cause suppression or enhancement of the signal. This is due in large part to the fact that the detection of MRR spectra is very high resolution and gives a large number of very specific lines for one compound even if that compound is an isomer or isotopologue or isotopomer of another compound. Furthermore, there are no suppression effects as there are in mass spectrometry.

[0058] There are many applications in which the capabilities of GC-MRR spectroscopy are extremely useful. These include metabolomics, natural product screening, and environmental analysis, among others. As an example, consider the following environmental degradation study:

[0059] Determination of changes in isotopic composition is used to evaluate the biotic and abiotic reactions of organic feed / 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 a broadly applicable approach to study chemical reactions in complex matrices and to better understand the degradation of contaminants. This degradation can occur via enzymatic pathways or by photochemical or organic reactions consisting of substitution, elimination, or electron transfer.

[0060] CSIA provides detailed insight into the degradation pathway by precisely targeting reactive atoms in a given trace contaminant molecule. Currently, these problems are addressed by isotope ratio mass spectrometry of carbon, hydrogen, and nitrogen, but isotope ratio mass spectrometry measurements of oxygen, sulfur, and chlorine are challenging. Similarly, PSIA examines intramolecular isotope changes. Traditionally, PSIA (for carbon) is performed by off-line conversion of functional groups to carbon dioxide, followed by GC-MS, or site-specific natural isotope fractionation-nuclear magnetic resonance (SNIF-NMR). NMR is a time-consuming and low-sensitivity technique. Thus, significant preconcentration of the trace compounds is required prior to NMR measurements. In contrast, GC-MRR can address these challenging cases of CSIA and PSIA without post-column conversion or purification.

[0061] One example of using GC-MRR to study environmental degradation involves selective biodepletion of low concentration samples of pyridine isotopologues. Although the microbial metabolism of pyridine and other organic bases has been widely studied, to our knowledge, no isotope-specific studies exist. Table 1 shows the isotope-specific depletion of pyridine isotopologues in three different microbial cultures / solutions. 14 N and 15 GC-MRR results for biodepletion of N-pyridine standards are shown. Both pyridine isotopologues were depleted over time in these cases. E. coli cultures were 14 shows selective depletion of N-pyridine isotopologues, whereas B. cepacia and groundwater show no significant differences. Dynamic isotope fractionation is well known for inorganic nitrogen sources and has been used to provide insight into the biological and enzymatic hydrolysis of herbicides.

[0062] Table 2 shows distinctly different kinetics of biodepletion for the various isotopologues of acetonitrile by two different bacteria. The data show distinct kinetics of biodepletion for the various isotopologues of acetonitrile by two different bacteria. The normal form of acetonitrile was depleted at the highest rate by E. coli, with no detectable differences among the three single-substituted isotopologues. More interestingly, in the case of V. fischeri, CH 313 The fact that CN isotopologues are selectively depleted. No other analytical method or combination of methods allows for such easy characterization of these phenomena. This type of extensive study of site-specific isotope analysis would be very informative for biodegradation studies and is currently less explored due to the lack of suitable analytical tools. GC-MRR solves this deficiency. [Table 1] [Table 2]

[0063] [Liquid chromatography (LC)-MRR spectroscopy] 9 shows a liquid chromatography (LC)-MRR spectroscopy system 900 that includes a liquid chromatograph 910 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 liquid analytes via inlet 916, for example, from a continuously flowing sample source. A solvent flowing from a solvent source 912 passes the liquid analytes through the column 914 where the analytes at least partially separate into their constituent components.

[0064] Volatilization interface 930 couples the at least partially separated analyte components to the MRR spectrometer 920. It volatilizes the analyte components - for example, the analyte components can be heated until they vaporize. A carrier gas, such as neon or helium from a carrier gas source 934, pushes the volatilized analytes through volatilization interface 930 and into a pulse-jet ultrasonic nozzle 932 that is coupled to volatilization interface 930. Nozzle 932 pushes the volatilized analyte component(s) through a vacuum pressure (e.g., 10 -6The microwaves are pumped down by a vacuum pump 925 to 1000 psi (2000 psi) to a measurement chamber 924 of the MRR spectrometer. A microwave source (not shown) as in FIG. 3A irradiates the analyte component(s) in the measurement chamber with one or more targeted or broadband excitation pulses. A receiver (not shown) as in FIG. 3A detects the FID signal emitted by the analyte component(s) volatilized in response to the excitation pulse(s). Electronics (not shown) detect and process the FID signal to produce an MRR spectrum. The MRR spectrum can be measured continuously or on demand, for example, in response to detection of a peak in the LC column output sensed by an auxiliary detector.

[0065] [Chiral analysis using GC-MRR spectroscopy] Chiral analysis is an area where GC-MRR can have significant added value, particularly in the conversion of enantiomers to diastereomers in MRR for chiral analysis using a gas-phase complexation technique called chiral tagging. A small number of small, volatile chiral molecules have been shown to efficiently complex with a wide range of chemical analytes, allowing accurate enantiomeric excess determination of these compounds in mixtures. For more information on chiral tagging, see, for example, U.S. Patent Application Publication No. 2019 / 0302015, entitled "Cavity-Enhanced Fourier Transform Spectroscopy for Chiral Analysis," which is incorporated herein by reference in its entirety.

[0066] Figures 10A and 10B show the idea behind chiral tagging. Briefly, chiral tagging involves "tagging" an analyte with a small chiral molecule of known stereochemistry, such as the verbenone / butynol system in Figure 10A. Weakly bound complexes, stabilized by a combination of hydrogen bonding, van der Waals, and other forces, can be efficiently formed with a pulsed ultrasonic expansion nozzle. The resulting diastereomeric complexes have different moments of inertia and can be analyzed by the capabilities of MRR spectroscopy, as described above and shown in Figure 10B. Chiral analysis is important in a wide range of applications, including pharmaceutical, environmental, and metabolite analysis.

[0067] More specifically, Figure 10A shows that the enantiomers of verbenone (an example of a chiral molecule) have the same moment of inertia and therefore the same MRR spectrum. However, when a chiral tag (in this case, (S)-3-butyn-2-ol) is complexed with the two enantiomers, a difference in the moment of inertia occurs, which results in two species with different moments of inertia and therefore different MRR spectra. The two complexes are called heterochiral (i.e., (R,R)-verbenone + (S)-3-butyn-2-ol) or homochiral (i.e., (S,S)-verbenone + (S)-3-butyn-2-ol).

[0068] Figure 10B shows two spectral lines, one each for the heterochiral and homochiral complexes of Figure 10A. In a racemic mixture of butynol tags, the two complexes are seen at approximately the same intensity (they differ due to complex dipole moments, formation ratios, and other factors). With pure (S)-3-butyn-2-ol as the tag, the lines are seen at different intensities. These line ratios can be used to calculate the enantiomeric ratio and / or enantiomeric excess of verbenone in the sample.

[0069] Chiral monitoring can be integrated into a GC-MRR instrument with an accessory system for introducing gas-phase chiral tags by an additional valve at the interface of the GC outlet, before the pulse jet sampling source. The tags can be mixed with the sample eluting post-column. The spectral library can contain entries of reference chiral molecules (chiral tags). This chiral MRR method can be used instead of or in combination with chiral GC separations to open the range of new separation and identification possibilities.

[0070] [Conclusion] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those 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 the actual parameters, dimensions, materials, and / or configurations will depend on the particular application for which the teachings of the present invention are used. Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed herein. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0071] Also, various inventive concepts may be embodied as one or more methods, examples of which have been presented. The acts performed as part of a method may be ordered in any suitable manner. Thus, embodiments may be constructed in which acts are performed in a different order than illustrated, and which may include performing some acts simultaneously even though shown as sequential acts in the exemplary embodiments.

[0072] All definitions defined and used herein are to be understood to supersede any dictionary definitions, definitions in any documents incorporated herein by reference, and / or ordinary meaning of the defined terms.

[0073] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated otherwise, should be understood to mean "at least one."

[0074] The term "and / or" as used in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjointly present in some cases and disjointly present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified in the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may in one embodiment refer to only A (which may optionally include elements other than B); in another embodiment, it may refer to only B (which may optionally include elements other than A); in yet another embodiment, it may refer to both A and B (which may optionally include other elements), etc.

[0075] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be construed as inclusive, i.e., including at least one, but also including a plurality of a number or list of elements, and optionally including additional unlisted items. Only when used in a term clearly indicating the contrary, such as "only one of" or "exactly one of," or in the claims, "consisting of" refers to including exactly one element of a number or list of elements. In general, the term "or" as used herein will only be construed as an exclusive alternative (i.e., "one or the other, but not both") when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, has its ordinary meaning as used in the field of patent law.

[0076] As used in this specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but need not include at least one of every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that there may optionally be elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified element. 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") could refer, in one embodiment, to at least one, and optionally more than one, A, with B absent (and optionally including elements other than B); in another embodiment, to at least one, and optionally more than one, B, with A absent (and optionally including elements other than A); in yet another embodiment, to at least one, and optionally more than one, A, and at least one, and optionally more than one, B (and optionally including other elements), etc.

[0077] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. As set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively.

Claims

1. a gas chromatograph for separating an analyte into a plurality of components, the gas chromatograph having a column in fluid communication with a carrier gas source that provides a carrier gas to propel the plurality of components through the column; a molecular rotational resonance (MRR) spectrometer configured to measure MRR spectra of the plurality of components, a measuring chamber for holding the plurality of components; a nozzle in fluid communication with the column and the measuring chamber for injecting the plurality of components into the measuring chamber; a microwave source in electromagnetic communication with the measurement chamber for exciting the plurality of components being measured with an excitation pulse having at least one spectral component within a frequency range of about 6 GHz to about 18 GHz; an MRR spectrometer comprising: a processor operatively coupled to the MRR spectrometer for analyzing the components of the analyte based on the MRR spectra of the components; Equipped with The integrated chromatography-spectroscopy system, wherein the processor is configured to analyze co-eluting components of the analyte based on the MRR spectra of the components.

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

3. the carrier gas source is a first carrier gas source, the carrier gas is a first carrier gas; 10. The integrated chromatography-spectroscopy system of claim 1, further comprising a second carrier source in fluid communication with the nozzle for providing a second carrier gas for injecting the component into the measurement chamber, the second carrier gas rotatingly cooling the component.

4. a gas chromatograph for separating an analyte into a plurality of components, the gas chromatograph having a column in fluid communication with a carrier gas source that provides a carrier gas to propel the plurality of components through the column; a molecular rotational resonance (MRR) spectrometer configured to measure MRR spectra of the plurality of components, a measuring chamber for holding the plurality of components; a nozzle in fluid communication with the column and the measuring chamber for injecting the plurality of components into the measuring chamber; a microwave source in electromagnetic communication with the measurement chamber for exciting the plurality of components being measured with an excitation pulse having at least one spectral component within a frequency range of about 6 GHz to about 18 GHz; an MRR spectrometer comprising: a processor operatively coupled to the MRR spectrometer for analyzing the components of the analyte based on the MRR spectra of the components; a chiral tag source in fluid communication with the nozzle for injecting a chiral tag into the measurement chamber containing the component, the chiral tag binding to a different enantiomer of at least one of the components, thereby producing a shift in the MRR spectrum of the different enantiomer that can be resolved by the MRR spectrometer; An integrated chromatography-spectroscopy system comprising:

5. a gas chromatograph for separating an analyte into a plurality of components, the gas chromatograph having a column in fluid communication with a carrier gas source that provides a carrier gas to propel the plurality of components through the column; a molecular rotational resonance (MRR) spectrometer configured to measure MRR spectra of the plurality of components, a measuring chamber for holding the plurality of components; a nozzle in fluid communication with the column and the measuring chamber for injecting the plurality of components into the measuring chamber; a microwave source in electromagnetic communication with the measurement chamber for exciting the plurality of components being measured with an excitation pulse having at least one spectral component within a frequency range of about 6 GHz to about 18 GHz; an MRR spectrometer comprising: a processor operatively coupled to the MRR spectrometer for analyzing the components of the analyte based on the MRR spectra of the components; an auxiliary detector operatively coupled to the column and to the processor for detecting peaks in the output of the column; Equipped with The integrated chromatography-spectroscopy system, wherein the processor is configured to trigger measurement of the MRR spectrum in response to the peak and / or to process the MRR spectrum based at least in part on the peak.

6. a gas chromatograph for separating an analyte into a plurality of components, the gas chromatograph having a column in fluid communication with a carrier gas source that provides a carrier gas to propel the plurality of components through the column; a molecular rotational resonance (MRR) spectrometer configured to measure MRR spectra of the plurality of components, a measuring chamber for holding the plurality of components; a nozzle in fluid communication with the column and the measuring chamber for injecting the plurality of components into the measuring chamber; a microwave source in electromagnetic communication with the measurement chamber for exciting the plurality of components being measured with an excitation pulse having at least one spectral component within a frequency range of about 6 GHz to about 18 GHz; an MRR spectrometer comprising: a processor operatively coupled to the MRR spectrometer for analyzing the components of the analyte based on the MRR spectra of the components; a display operatively coupled to said processor for displaying a relationship of the peaks in the output of said column to a plurality of MRR spectra; An integrated chromatography-spectroscopy system comprising:

7. a chromatograph for separating an analyte into a plurality of components, the plurality of components including co-eluting components that cannot be separated by the chromatograph; a molecular rotational resonance (MRR) spectrometer in fluid communication with the chromatograph for measuring a molecular rotational resonance (MRR) spectrum of at least one of the plurality of components; a processor operatively coupled to the MRR spectrometer for analyzing co-eluting components from the chromatograph based at least in part on the MRR spectrum; A system comprising:

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

9. the MRR spectrometer being a microwave MRR spectrometer having an expansion chamber; 10. The system of claim 8, further comprising a carrier gas source in fluid communication with the gas chromatograph for passing the analyte through the gas chromatograph and for forcing at least one of the plurality of 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 MRR 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 MRR spectrometer being a microwave MRR spectrometer having an expansion chamber, the system comprising: a volatilization interface in thermal communication with the liquid chromatograph for volatilizing at least one of the plurality of components; a carrier gas source in fluid communication with the volatilization interface for forcing at least one of the plurality of components through the volatilization interface and into the expansion chamber; The system of claim 12 further comprising:

14. 8. The system of claim 7, wherein the MRR spectrometer is configured to measure a spectrum having a bandwidth of at least 50 MHz of at least one co-eluting component exiting the chromatograph.

15. 8. The system of claim 7, further comprising a processor operatively coupled to the MRR spectrometer for forming a total molecular chromatogram of the analyte based at least in part on the MRR spectra of the plurality of components.

16. 8. The system of claim 7, further comprising a processor operatively coupled to the MRR spectrometer for resolving at least one of isomers, isotopologues, or isotopomers from the plurality of components of the analyte based at least in part on the MRR spectrum.

17. a chromatograph for separating an analyte into a plurality of components, the plurality of components including co-eluting components that cannot be separated by the chromatograph; a molecular rotational resonance (MRR) spectrometer in fluid communication with the chromatograph for measuring a molecular rotational resonance (MRR) spectrum of at least one of the plurality of components; an auxiliary detector operatively connected to the chromatograph for detecting peaks in the output of the chromatograph; a processor operatively coupled to the MRR spectrometer and the auxiliary detector for triggering measurement of the MRR spectrum in response to the peak and / or for processing the MRR spectrum based at least in part on the peak; A system comprising:

18. 8. The system of claim 7, further comprising a nozzle in fluid communication with the chromatograph and the MRR spectrometer for rotationally cooling molecules of at least one of the plurality of components before the MRR spectrometer measures the MRR spectrum.

19. 1. A method for analyzing an analyte, comprising: chromatographically separating the analyte into a plurality of components, the plurality of components including co-eluting components that cannot be separated by the chromatograph; measuring a molecular rotational resonance (MRR) spectrum of at least one of the components with a molecular rotational resonance (MRR) spectrometer in fluid communication with the chromatograph; analyzing at least one of isomers, isotopologues, or isotopomers from among a plurality of components of the analyte based at least in part on the MRR spectrum; A method comprising:

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

21. detecting peaks in the output of the chromatograph; triggering a measurement of the MRR spectrum in response to detecting the peak; 21. The method of claim 20, further comprising:

22. 20. The method of claim 19, wherein the step of measuring an MRR spectrum of at least one of the plurality of components comprises measuring a broadband spectrum of an effluent exiting the chromatograph.

23. and rotating cooling the molecules of at least one of the plurality of components prior to measuring the MRR spectrum of at least one of the plurality of components; 20. The method of claim 19, wherein measuring the MRR spectrum comprises exciting at least one of the plurality of components with a microwave excitation signal.

24. 20. The method of claim 19, further comprising identifying at least one unknown component of the analyte based at least in part on the MRR spectrum.

25. 20. The method of claim 19, further comprising quantifying said at least one of said components based at least in part on said MRR spectrum.

26. 20. The method of claim 19, further comprising forming a total molecular chromatogram of the analyte based at least in part on the MRR spectrum.

27. the chromatograph is a gas chromatograph and the MRR spectrometer is a microwave MRR spectrometer having an expansion chamber; 20. The method of claim 19, further comprising passing the analyte through the gas chromatograph and forcing at least one of the plurality of components into the expansion chamber with the carrier gas.

28. the chromatograph is a liquid chromatograph and the MRR spectrometer is a microwave MRR spectrometer having an expansion chamber; volatilizing at least one of the plurality of components at a volatilization interface; 20. The method of claim 19, further comprising using a carrier gas to push at least one of the components away from the volatilization interface and into the expansion chamber.

29. 1. A method for analyzing an analyte, comprising: chromatographically separating the analyte into a plurality of components, the plurality of components including co-eluting components that cannot be separated by the chromatograph; measuring a molecular rotational resonance (MRR) spectrum of at least one of the components with a molecular rotational resonance (MRR) spectrometer in fluid communication with the chromatograph; attaching a chiral tag to at least one of the plurality of components prior to measuring the MRR spectrum; identifying an enantiomer among the components of the analyte based on the MRR spectrum and / or determining the enantiomeric excess of the components of the analyte based on the MRR spectrum; A method comprising:

30. a gas chromatograph for separating an analyte into a plurality of components; a molecular rotational resonance (MRR) spectrometer in fluid communication with the gas chromatograph for measuring MRR spectra of the plurality of components; a pulse jet ultrasonic expansion source in fluid communication with a column of the gas chromatograph and a vacuum chamber of the MRR spectrometer for transporting the components from the column to the vacuum chamber; Equipped with The system, wherein the MRR spectrometer is configured to analyze isomeric compounds by MRR spectroscopy.

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

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

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

34. 31. The system of claim 30, wherein the MRR spectrometer is configured to analyze co-eluted components by the gas chromatograph.

35. a gas chromatograph for separating an analyte into a plurality of components; a molecular rotational resonance (MRR) spectrometer in fluid communication with the gas chromatograph for measuring MRR spectra of the plurality of components; a pulse jet ultrasonic expansion source in fluid communication with a column of the gas chromatograph and a vacuum chamber of the MRR spectrometer for transporting the components from the column to the vacuum chamber; A spectroscopic system comprising: The spectroscopic system is configured to identify and quantify the components without a reference standard.

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