Molecular rotational resonance spectrometer for synchronous measurements and method of use thereof

Abstract

Molecular rotational resonance (MRR) spectroscopy is a structurally specific, high-resolution spectroscopic technique capable of identifying chemical species, including enantiomers, that cannot be resolved by other analytical techniques. In MRR spectroscopy, a gas-phase sample is injected into a vacuum chamber, where it is excited with millimeter-wave or microwave radiation and emits a free induction pulse (FID) in response. The spectrum of the FID pulse provides information about the sample's chemical structure. In MRR ratio measurements, the sample is simultaneously illuminated by two excitation pulses at different frequencies (e.g., a broadband pulse and a narrowband pulse at different frequencies, or a narrowband pulse at different frequencies). The pulses stimulate simultaneous or overlapping FID emissions from different components of the sample. Comparing the spectra of these FID emissions yields the ratio of the components in the sample.

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. patent application Ser. No. 63 / 486,235, filed February 21, 2023, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Molecular rotational resonance (MRR) spectroscopy (also known as molecular rotational spectroscopy or microwave spectroscopy) identifies and characterizes molecules, including compounds, by exciting molecules with electromagnetic radiation based on their characteristic rotational angular momenta and measuring the coherent radiation emitted by the molecules as a result of the excitation via free induction decay (FID). The emitted radiation is detected as a time-varying signal. Fourier transforming the signal produces a spectrum that can be used to identify the characteristic rotational angular momenta of molecules in a sample, which may then be used to identify and / or quantify analytes and their components.

[0003] The energy levels of a molecule are quantified according to the three-dimensional mass distribution of the molecule, which is expressed as the moment of inertia, I, which is given by I=Σm i r i 2 is defined (in one dimension) 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 on its moments of inertia in the three spatial axes. By using rotational spectroscopy, molecules can be clearly distinguished by their differences in structure, due to their numerous very narrow spectral lines (typically with a spectral resolution of n / Dn ≈ 10 -5), high-resolution rotational spectra are unique to each molecular structure.

[0004] MRR spectroscopy is typically performed in a vacuum chamber that provides a low-pressure environment (e.g., less than 100 mTorr) and uses electromagnetic radiation in the microwave to millimeter-wave spectrum (e.g., 1-40 GHz in the microwave region and 30-3000 GHz in the millimeter region) for excitation. The extremely fine resolution of MRR spectroscopy means that the patterns (spectra) of different molecules can be directly resolved in an unseparated mixture. Furthermore, the structure of the patterns depends only on the three-dimensional structure (mass distribution and electronic charge distribution) of the molecules, which can be accurately and efficiently calculated by commercially available quantum chemistry software. With MRR spectroscopy, molecules can be directly identified in complex mixtures without the need for pure standard samples, which can be very expensive and difficult to generate.

[0005] For example, Figure 1 13 CH3CN and CH3 13 The MRR spectrum of CN shows isotopomers (isotopes) with identical masses, and each spectral pattern can be clearly separated. In particular, the MRR spectrum of each molecule can be calculated with high accuracy. Therefore, it is possible to unambiguously identify molecules using MRR without a standard sample. Summary of the Invention

[0006] MRR spectroscopy is typically performed by one of two methods: broadband or targeted. Broadband measurements are performed by exciting an analyte sample with one or more broadband pulses of electromagnetic radiation (e.g., chirped pulses of microwave or millimeter-wave radiation). Compared to targeted measurements, broadband measurements can acquire a spectrum of the sample with a relatively large bandwidth (e.g., 5 MHz or greater) at the expense of reduced sensitivity and / or spectral resolution. Broadband measurements are often used to characterize most, or even all, of the components in a sample (e.g., a mixture) containing unknown or unexpected molecules.

[0007] Targeted measurements are performed by exciting a sample with narrowband pulses of electromagnetic radiation (e.g., pulses of microwave or millimeter-wave radiation resonant with the cavity containing the sample). Compared to broadband measurements, the spectrum of the sample acquired in targeted measurements has a relatively small bandwidth (e.g., 1 MHz or less), which can provide greater sensitivity (e.g., 10-100 times) due to the excitation power being concentrated in a smaller frequency range for the same measurement time and / or greater spectral resolution. Targeted measurements are often used to quantify and / or detect known components (e.g., chemical species) in a sample.

[0008] MRR spectroscopy can provide sufficient sensitivity to distinguish individual components in a sample (e.g., isotopomers) and / or quantify trace amounts of specific components. Thus, in principle, multiple MRR measurements can be used to distinguish and compare the relative abundances of different chemical species within or between different samples, particularly for species that are difficult to distinguish and / or quantify using other analytical chemistry techniques. For example, broadband and targeted MRR measurements can provide complementary information about a sample. Broadband MRR measurements can detect and monitor most, or in some cases, all, of the known and unknown components in a sample, while targeted MRR measurements can selectively detect and quantify known components with greater sensitivity and lower detection limits. In another example, a first targeted MRR measurement can detect and quantify a first component in a sample, and a second targeted MRR measurement can detect and quantify a second component in the same sample to compare the relative amounts of the first and second components.

[0009] However, conventional MRR spectrometers or MRR spectroscopy systems generally can perform only one measurement on a sample at a time. In other words, conventional MRR spectrometers cannot simultaneously perform multiple broadband and / or targeted measurements on the same sample. As a result, various factors affecting measurement reproducibility, such as sample-to-sample variations or fluctuations in environmental conditions over time, subject data obtained from multiple MRR measurements to greater measurement error and loss of precision, making any quantitative analysis impossible. For example, conventional MRR spectroscopy is often performed by injecting a controlled amount of sample (also referred to herein as a "sample pulse") into a vacuum chamber. However, even when multiple sample pulses originating from the same sample source are injected using the same inlet, the composition of the sample pulse can vary significantly. In some cases, the total number density of molecules can vary by at least 10% between different sample pulses. In another example, MRR spectrometers are typically susceptible to long-term drift due to thermal effects in the spectrometer and / or changes in the sample, chamber vacuum level, and / or pulse nozzle over time (e.g., due to changes in solenoid characteristics or poppet wear). Therefore, multiple MRR measurements acquired over an extended period (e.g., a 24-hour period) may yield different results, even when the same sample is being characterized.

[0010] Accordingly, the present disclosure is directed to various inventive MRR spectrometers capable of simultaneously performing multiple synchronous measurements on the same sample. The synchronous measurements may include, for example, a combination of at least one broadband measurement and at least one targeted measurement, or a combination of two or more targeted measurements. The MRR spectrometer includes a vacuum chamber for holding the sample during measurements. The vacuum chamber provides a low-vacuum environment, generated in part by a vacuum pump and measured by a pressure gauge operably connected to the vacuum chamber.

[0011] In one non-limiting example, the MRR spectrometer may include a broadband spectrometer and a target spectrometer for performing broadband and target measurements on a sample, respectively. The broadband spectrometer may include several components disposed within a vacuum chamber, such as a source horn antenna for transmitting broadband pulses of electromagnetic radiation (e.g., chirped pulses of microwave or millimeter-wave radiation) and a receiver horn antenna for receiving electromagnetic radiation emitted by components in the sample in response to the broadband pulses. The target spectrometer may include several components disposed within the vacuum chamber, such as a fixed mirror and a movable mirror with a translation stage that together form an adjustable Fabry-Perot cavity, a source antenna for transmitting narrowband pulses of electromagnetic radiation, and a receiver antenna for receiving electromagnetic radiation emitted by components in the sample in response to the narrowband pulses. The MRR spectrometer may further include one or more signal generators for generating broadband and / or narrowband pulses, and analog-to-digital converters (ADCs) or digitizers for receiving and converting the emitted radiation received by the receiver antennas of the broadband and target spectrometers for subsequent processing and analysis. Other inventive MRR spectrometers may include different combinations of spectrometers, for example, two target spectrometers.

[0012] The MRR spectrometer of the present invention may further include at least one sampling inlet for injecting a sample of analyte into the vacuum chamber. The sampling inlet may operate in several ways. In one example, the sampling inlet may be connected to a pulsed valve driver to controllably inject sample pulses into the vacuum chamber at desired time intervals. The sample pulses may propagate through the vacuum chamber before being removed by a vacuum pump. In another example, the sampling inlet may inject the sample into the vacuum chamber as a continuous stream, and the vacuum pump may continuously remove the sample so that the sample does not accumulate in the vacuum chamber. In some examples, multiple sampling inlets may be used to selectively inject sample and standard samples into the chamber for measurement.

[0013] In one non-limiting example, the sampling inlet may be a pinhole nozzle that injects the sample along with a carrier gas as particulates that are maintained in the gas phase. The sampling inlet may also be connected to a sample source. The sample source may include, for example, a reservoir that receives the analyte sample from a flow reactor or syringe, and a carrier gas source that provides a flow of carrier gas to facilitate injection of the sample through the nozzle and / or to purge various fluid lines after each measurement. The sample source may further include a gas or liquid chromatograph for separating individual components of the sample prior to injection into the vacuum chamber.

[0014] The way in which multiple MRR measurements are simultaneous or considered simultaneous can vary depending on how the sample is injected into the vacuum chamber.

[0015] When a sampling inlet injects a sample into a vacuum chamber as a series of pulses (also referred to herein as "sample pulses"), multiple MRR measurements can be considered simultaneous if each measurement is performed with the same sample pulse. For example, the source antennas of the broadband spectrometer and the target spectrometer may emit broadband and narrowband pulses that irradiate the same sample pulse, and the receiver antennas of the broadband and target spectrometers may detect FID emissions from the same sample pulse in response to the broadband and narrowband pulses.

[0016] When a sampling inlet injects a sample into a vacuum chamber as a continuous stream, multiple MRR measurements are considered simultaneous if each measurement is performed on similar or identical portions of the sample stream within a sufficiently short period of time so that the composition of the portion of the sample stream being characterized remains substantially the same. For example, a source antenna of a broadband spectrometer may emit a broadband pulse to irradiate a first portion of the sample stream, and a source antenna of a target spectrometer may emit a broadband pulse to irradiate a second portion of the sample stream. The second portion of the sample stream should substantially overlap with the first portion of the sample stream (e.g., they may overlap by 50%, 60%, 70%, 80%, 90%, or more). Furthermore, both the broadband and target MRR measurements should be performed within a predetermined period of time (e.g., 1 millisecond or less).

[0017] Generally, a sample pulse or portion of a sample stream can be exposed to a broadband and narrowband pulse, which may be significantly longer than the FID phase relaxation time (e.g., the sample pulse may have a duration of 1-4 μs, while the FID phase relaxation time may be in the range of 10-40 μs). In some instances, the MRR spectrometer may probe a particular sample pulse or portion of the sample stream using multiple broadband and / or narrowband pulses and detect corresponding FID emissions in response to the broadband and / or narrowband pulses. While the MRR spectrometers disclosed herein are capable of performing multiple MRR measurements simultaneously, it should be understood that individual MRR measurements (e.g., a single broadband MRR measurement, a single target MRR measurement) can be performed separately on the sample, as desired.

[0018] The MRR spectrometer further includes a timing controller for synchronizing the operation of at least a plurality of spectrometers and the sampling inlet (e.g., via pulsed valve drivers). For example, the timing controller may control when the target MRR spectrometer and the broadband MRR spectrometer generate their respective excitation pulses for measurement, when the detectors (e.g., receiver antennas and ADCs) receive and record signals (e.g., FID emission signals) generated by the excitation pulses interacting with the sample, and when the sampling inlet injects sample pulses into the system (e.g., relative to the generation of excitation pulses and / or reception of signals by the detectors). This may be accomplished, for example, by a timing controller including a frequency standard (e.g., a rubidium atomic clock) that provides a reference clock signal for synchronizing the operation of components within the MRR spectrometer.

[0019] When multiple MRR measurements are performed simultaneously, multiple pulses (e.g., broadband pulses, narrowband pulses) and FID emissions may be present in the vacuum chamber at the same time, which may generate additional noise for the measurements. In some instances, for example, to reduce or mitigate noise that may be generated by simultaneously performing broadband and target measurements, components of the spectrometers may be arranged to reduce the amount of electromagnetic radiation associated with one spectrometer that reaches the detector of another spectrometer.

[0020] In one non-limiting example, the vacuum chamber may be shaped as a six-way cross structure with a first axis of the broadband spectrometer, a second axis of the target spectrometer, and a third axis of various other hardware of the MRR spectrometer (e.g., ports for connecting to vacuum pumps, pressure gauges, and / or other equipment to facilitate operation of the MRR spectrometer). The first, second, and third axes may be orthogonal to one another. Generally, FID emissions occur in a similar direction to the excitation pulses that generate them. By arranging the components of the broadband spectrometer and the target spectrometer on different axes, FID emissions associated with broadband pulses are less likely to propagate or travel along the axis of the target spectrometer, and FID emissions associated with narrowband pulses are less likely to propagate or travel along the axis of the broadband spectrometer. Additionally, RF absorbing material may be placed on the interior walls of the chamber to reduce the effects of cavity rings, particularly at frequencies probed by the broadband MRR spectrometer.

[0021] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (unless those concepts are mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should be understood that terms explicitly used herein that may also appear in any disclosure made a part of this specification by reference should be given the meaning most consistent with the particular concepts disclosed herein. [Brief explanation of the drawings]

[0022] Those skilled in the art will understand 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, and in some instances, various aspects of the inventive subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of different features. In the drawings, like reference characters generally refer to like features (e.g., functionally similar and / or structurally similar elements).

[0023] [Figure 1] FIG. 1 shows the molecular rotational resonance (MRR) spectra of two isotopomers of acetonitrile, demonstrating very high resolution of the MRR spectral lines along with selectivity for isobaric compounds. [Figure 2] FIG. 2 shows a block diagram of an MRR measurement device of the present invention configured for simultaneous broadband and targeted measurements. [Figure 3] FIG. 3 shows a three-port sampling interface suitable for use in the MRR spectrometer of the present invention. [Figure 4A] FIG. 4A shows the front end of an MRR spectrometer of the present invention with separate channels and analog-to-digital converters (ADCs) for the broadband and target signal channels. [Figure 4B] FIG. 4B shows the front end of an MRR spectrometer of the present invention with frequency multiplexed channels and a single ADC for the broadband and target signal channels. [Figure 5A] FIG. 5A shows a schematic diagram of an MRR spectrometer of the present invention configured to perform simultaneous broadband and targeted measurements of an analyte gas sample. [Figure 5B] FIG. 5B shows an MRR ratiometer instrument with two Balle-Flygare type target spectrometers arranged in orthogonal cavities and a single nozzle. [Figure 5C] FIG. 5C shows an MRR ratiometer instrument with two Balle-Flygare type target spectrometers arranged with orthogonal cavities and a pair of symmetric nozzles. [Figure 5D] Figure 5D shows a dual-polarization MRR ratiometer with two movable flat reflectors (metal mirror and wire grid polarizer) and two pulsed nozzle sources for sample injection into the vacuum cavity of the MRR ratiometer of the same volume. [Figure 5E] Figure 5E shows a folded-cavity MRR ratiometer with a wire-grid polarizing beam splitter fixed within a confocal microwave cavity and a side-mounted nozzle. [Figure 5F]Figure 5F shows a folded-cavity MRR ratiometer with a wire-grid polarizing beam splitter fixed within a confocal microwave cavity and a coaxially mounted nozzle. [Figure 5G] Figure 5G shows an MRR ratiometer with a dual-polarization, cavity-enhanced detection system (target spectrometer) with a fixed flat mirror and a movable spherical mirror arranged in a semi-confocal mirror geometry. [Figure 5H] Figure 5H shows an MRR ratiometer with a dual-polarization cavity-enhanced detection system (target spectrometer) with a fixed spherical mirror and a movable flat mirror arranged in a semi-confocal mirror geometry. [Figure 6A] FIG. 6A shows a flowchart of an exemplary method for simultaneously performing broadband and target measurements. [Figure 6B] FIG. 6B shows an exemplary timing diagram of an analyte sample pulse, a broadband excitation pulse from the broadband spectrometer, and a target excitation pulse from the target spectrometer. [Figure 7A] FIG. 7A illustrates chiral tagging, which is the gas phase formation of different diastereomers with different spectra by combining a chiral tag and an enantiomer. [Figure 7B] Figure 7B shows the enantiomeric excess analysis from MRR spectroscopy. Measurements were performed using either the racemic or pure enantiomers of the chiral tag. [Figure 8] FIG. 8 is a calibration curve for determining the enantiomeric excess of 1,1,1-trifluoropropan-2-ol (TFIP) using chiral tag rotational spectroscopy. [Figure 9A] Figure 9A shows the signal fluctuations of individual MRR spectroscopic transitions observed in TFIP measurements. [Figure 9B] Figure 9B shows signal fluctuations or variations in back-to-back measurements of TFIP dimer transitions. [Figure 9C] Figure 9C shows the signal fluctuations or variations for the transition of TFIP monomer. [Figure 10]FIG. 10 shows the variation in the ratio of transition intensities of the 13C isotopomers and the 12C "normal species" of TFIP in the monomer spectrum. [Figure 11] FIG. 11 shows MRR ratiometric measurements of 13C:12C isotope signal ratios in TFIP. [Figure 12] FIG. 12 shows 60 consecutive measurements of the 13C:12C ratio in a TFIP sample made using the MRR ratiometer of FIG. 5A. [Figure 13] FIG. 13 is a graph of the difference between successive measurements in the series of 60 consecutive measurements shown in FIG. [Figure 14] FIG. 14 is a plot showing the enantiomeric excess (EE) measurement results of a highly enantiomeric pure sample of (R)-TFIP measured using the MRR ratiometer of FIG. 5A. DETAILED DESCRIPTION OF THE INVENTION

[0024] Below is a more detailed description of various concepts and implementations related to an MRR spectrometer capable of simultaneously performing multiple MRR measurements. Multiple MRR measurements include, for example, a combination of at least one broadband MRR measurement and at least one targeted MRR measurement, or a combination of two or more targeted MRR measurements. The various concepts introduced above and discussed in more detail below can be implemented in multiple ways. Examples of specific implementations and applications are provided primarily for illustrative purposes so that those skilled in the art can implement implementations and alternatives that will be apparent to those skilled in the art.

[0025] The figures and exemplary implementations described below are not intended to limit the scope of the implementation to a single embodiment. Other implementations are possible by replacing some or all of the elements described or illustrated. Furthermore, when certain elements of the exemplary implementations of the present disclosure can be implemented partially or completely using known components, in some instances, only the portions of such known components necessary for understanding the implementation are described, and detailed descriptions of other portions of such known components are omitted so as not to obscure the implementation.

[0026] In the following discussion, various embodiments of an MRR spectrometer of the present invention are provided, with a given embodiment or set of embodiments illustrating one or more particular features of a vacuum chamber, a broadband spectrometer, a target spectrometer, a sampling inlet, timing circuitry, and various hardware to facilitate operation of the aforementioned components, such as hardware to operate the sampling inlet or move a movable mirror in the target spectrometer. Features discussed in connection with a given embodiment of an MRR spectrometer may be employed in other embodiments of an MRR spectrometer according to the present disclosure (provided that the respective features are not incompatible with one another), such that the various features disclosed herein may be readily combined in a given MRR spectrometer according to the present disclosure.

[0027] Certain dimensions and features of the MRR spectrometer are described herein using the terms "approximately," "about," "substantially," and / or "similar." As used herein, the terms "approximately," "about," "substantially," and / or "similar" indicate that each described dimension or feature is not a precise boundary or parameter and does not exclude functionally similar variations therefrom. Unless the context or description indicates otherwise, use of the terms "approximately," "about," "substantially," and / or "similar" in connection with a numerical parameter indicates that the numerical parameter includes variations that do not alter the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.).

[0028] (1.MRR ratio meter) The MRR ratiometer of the present invention can perform two simultaneous Fourier transform microwave (FTMW) spectroscopy measurements on the same sample. These measurements may be performed on different molecular components or species of the sample, which may be a mixture of different compounds or components, and can be used to determine relative representations of the components, such as the number density ratio or mass ratio of the components. Both measurements can be targeted (narrowband) measurements, or one measurement can be targeted and the other broadband. In either case, the resonant frequencies of the two species being measured should be different, allowing for simultaneous measurement of both species in the same sample volume. Simultaneous measurement of two chemically distinct species eliminates signal variations caused by variations in the number of molecules injected by the pulsed nozzle source in each measurement cycle.

[0029] In the MRR measurement device of the present invention, at least one of the FTMW spectroscopy measurements can be performed using cavity-enhanced FTMW spectroscopy. The increased sensitivity of cavity-enhanced FTMW spectroscopy shortens the time required to acquire signals of lower abundance molecular species down to a specified detection limit. The cavity can be either confocal or semi-confocal.

[0030] The MRR ratiometer of the present invention may also have the ability to inject two different samples, an analyte and a standard sample, into the same spatial region of the spectrometer's vacuum cavity at different times. The standard sample provides a comparison with the analyte for calibrating the measurement. The analyte sample and the standard sample can each be introduced separately into the vacuum cavity by pulse-jet expansion of a diluted sample mixture in an inert gas (typically 0.1% of the molecular sample diluted with neon). Furthermore, by switching between the standard and analyte samples over the course of the measurement (e.g., once per second or faster), long-term drift in the ratio determination can be reduced or eliminated. For example, the MRR measurement device of the present invention can switch between the standard and analyte samples on a pulse-to-pulse basis at a 10 Hz pulse rate (5 Hz for the reference pulse and 5 Hz for the analyte pulse).

[0031] For example, in stable isotope measurements to verify the authenticity of a sample, the standard sample may be a sample with a known ratio for a given pair of species, where a difference in the ratio of that pair of species in the analyte may indicate a forgery. 12 C isotope, and one of the carbon atoms in the molecule is 13 Consider monosubstituted isotopomers where C is substituted.

[0032] In enantiomeric excess measurements using chiral tag rotational spectroscopy, the standard sample may be a mixture of chiral samples studied with a racemic sample of the tag, and the analyte may be a mixture of samples studied with a highly enantiomeric pure sample of the tag. In this case, the monitored species may be homochiral and heterochiral complexes formed between the chiral sample and the tag in the pulse-jet expansion.

[0033] The MRR ratiometer of the present invention may include a pulse generation system capable of generating two microwave pulses at different frequencies substantially simultaneously. These pulses are applied to different sample components to be measured, with each pulse resonating with a selected rotational transition of a different molecular species. (When performing simultaneous broadband and targeted measurements, the broadband pulse may resonate with two or more species, and the targeted pulse may resonate with only one of those species or a different species.) In some cases, the pulse generation system uses microwave amplifiers to achieve sufficient pulse power to optimally excite the sample—the so-called π / 2 pulse condition in coherent excitation measurements. These amplifiers may be temperature stabilized to reduce long-term drift in pulse power and amplifier gain, which can cause fluctuations in the signals from the reference and analyte.

[0034] The MRR ratiometer of the present invention may include a coherent detection system that measures FID signals from both the standard and analyte samples. The amplitude of the oscillating electric field within the FID, which can be obtained by Fourier transform of the FID, is proportional to the number density of the monitored molecular species. The electric field amplitude of the FID signal is used to determine a relative indication, such as the number density ratio or mass ratio, of the two distinct molecular species being measured. The detection system may record FIDs from the two distinct molecular species using separate receiver and digitizer systems. As is common in FTMW spectroscopy, the receiver design may employ frequency downconversion using a mixer and a local oscillator (LO) signal to convert the signal to a lower frequency in the digitizer. The receiver typically uses low-noise microwave amplifiers in signal processing. These may be temperature stabilized to reduce amplifier gain fluctuations.

[0035] For high-precision measurements, the signals from the two molecular species being measured may be combined using a microwave voltage divider / combiner circuit element (such as a Wilkinson power divider) before any subsequent signal processing (signal amplification and / or frequency conversion). Combining the signals in the microwave domain can reduce variability in receiver performance for detecting two separate molecular signals at different resonant frequencies by providing an identical signal path through the receiver (amplifier, frequency conversion components, and digitizer) for both measured frequencies.

[0036] The ability of the MRR ratiometer of the present invention to simultaneously perform multiple MRR measurements can provide considerable benefits for a variety of applications. In one example, a broadband measurement can identify new compounds in a sample, and a targeted measurement can quantify the abundance of each new compound. In another example, multiple MRR measurements can be performed to compare the relative abundances of different compounds in a sample. For example, the high sensitivity of a targeted measurement can be utilized to quantify the amount of a first trace component in a sample. A separate targeted measurement (e.g., via a second targeted spectrometer) can be performed to quantify the amount of a second trace component in the sample. Alternatively, a broadband measurement can be used to quantify the amount of a second component in a sample if the second compound is more abundant and / or produces a stronger FID emission signal.

[0037] In another example, the MRR ratiometer of the present invention may be used to characterize isotopically labeled compounds. Given knowledge of the parent structure of the compound and / or where the label is located in the sample, broadband measurements may be used to identify isotopic variants of the compound in the sample, and targeted measurements may be used to quantify the amount of each isotopic variant, thus providing a distribution of isotopic variants present in the sample. Further details regarding the use of the MRR ratiometer of the present invention can also be found below.

[0038] In another example, the MRR ratiometer of the present invention can be used in high-throughput screening applications. For example, a collection of samples obtained by the same chemical reaction but under different experimental conditions can be characterized to determine which sample, and therefore the experimental conditions, provide the highest yield and / or higher conversion efficiency. Broadband measurements can be used to screen for unexpected impurities in each sample, and targeted measurements can be used to quantify the components of the sample and determine product yield.

[0039] In another example, the MRR ratiometer of the present invention may be used for reaction monitoring. For example, a sampling inlet of the MRR ratiometer of the present invention may receive a sample of the reaction mixture from a flow reactor. As the reaction occurs, the MRR ratiometer of the present invention can monitor and quantify various components of the reaction mixture to assess reaction completion, reaction kinetics, and / or impurity formation. Broadband measurements may be used to screen for unexpected impurities in each sample, and targeted measurements may be used to quantify components of the sample to determine product yield.

[0040] In another example, the MRR ratiometer of the present invention can be used to assess the purity of raw materials in a sample. Broadband measurements may be used to monitor the primary species of the sample (e.g., species that remain if the sample is pure), and targeted measurements may be used to identify and quantify known impurities in the sample.

[0041] In another example, the MRR ratiometer of the present invention may be used to analyze complex mixtures. Given a batch sample of a mixture, the MRR ratiometer of the present invention may identify and / or quantify components of the sample to assess batch-to-batch consistency of the mixture. Broadband measurements may be used to determine new, unknown compounds present in the sample, and targeted measurements may be used to quantify the amount of compounds present.

[0042] 2. MRR Spectrometer for Simultaneous Broadband and Targeted Measurements FIG. 2 shows a block diagram of an exemplary MRR spectrometer 200 capable of simultaneously performing broadband and targeted measurements. As shown, the MRR spectrometer 200 includes a vacuum chamber 202 for holding a sample (e.g., a sample pulse of an analyte, a continuous stream of sample), a pumping system 204 operably connected to the vacuum chamber 202 for generating and maintaining a low-vacuum environment within the vacuum chamber 202, and a sampling inlet 206 (e.g., a nozzle) connected to a sample source (not shown) for injecting the sample into the vacuum chamber 202. The pumping system 204 may include a vacuum pump, a pressure gauge, and / or a controller for managing the operation of the vacuum pump and the pressure gauge. The sampling inlet 206 may be connected to a pulse valve driver 208, which may be used to controllably inject sample pulses into the vacuum chamber 202 at desired time intervals (e.g., FIG. 6). The sampling inlet 206 may also inject a continuous stream of sample into the vacuum chamber.

[0043] The MRR spectrometer 200 is operably connected to the vacuum chamber 202 and includes both a target spectrometer 220 and a broadband spectrometer 230, which respectively perform (substantially) simultaneous target and broadband measurements on the same sample. The broadband spectrometer 230 may include two horn antennas 232 within the vacuum chamber 202: a source antenna that emits broadband pulses (e.g., chirp pulses) for excitation and a receiver antenna that receives FID emissions from the sample in response to the broadband pulses (see, e.g., the source and receiver horn antennas in FIG. 3 ). The target spectrometer 220 may also include two antennas within the vacuum chamber 202: a source antenna that emits narrowband pulses (e.g., resonant pulses) for excitation and a receiver antenna that receives FID emissions from the sample in response to the narrowband pulses (see, e.g., the source and receiver horn antennas in FIG. 3 ). The target spectrometer 220 may also include a pair of mirrors forming a Fabry-Perot cavity 222 to amplify the excitation of the sample by the resonant pulse and the resulting FID emission. One of the mirrors may be further movable as desired to facilitate adjustment of the resonant frequency of the Fabry-Perot cavity 222, for example, to correspond to a desired frequency for the target measurement. For example, the movable mirror may be coupled to a translation stage, as described below. Other inventive MRR spectrometers may include two target MRR spectrometers.

[0044] MRR spectrometer 200 includes hardware control circuitry and / or electronics 210 for controlling the operation of, for example, sampling inlet 206, target spectrometer 220, and / or broadband spectrometer 230 within MRR spectrometer 200. In one embodiment, hardware control circuitry 210 may set and maintain a desired sample rate injected into vacuum chamber 202 via sampling inlet 206 and / or timing intervals between sample pulses if pulsed operation is used. In another embodiment, hardware control circuitry 210 may adjust the position of a movable mirror within target spectrometer 220 via the translation stage described above.

[0045] MRR spectrometer 200 also includes timing control circuitry and / or electronics 212 for synchronizing the operation of at least sampling inlet 206, target spectrometer 220, and broadband spectrometer 230. Timing control circuitry 212 can ensure that broadband and target measurements are performed simultaneously on the same sample. Further aspects of timing control are discussed in more detail below.

[0046] MRR spectrometer 200 also includes a processor 216, such as a field-programmable gate array (FPGA), operably connected to hardware control circuitry 210, timing control circuitry 216, target spectrometer 220, broadband spectrometer 230, and pumping system 204. Processor 216 may also receive FID signals detected by receiver antennas of target and broadband spectrometers 220, 230. These FID signals may be converted from analog to digital signals by one or more digitizers 214 before transmitting to processor 216. In one non-limiting example, processor 216 may include or be operably connected to a display device (e.g., a computer monitor) for rendering and displaying a graphical user interface and at least one user input device (e.g., a mouse and / or keyboard) for a user to interact with the graphical user interface. For example, the graphical user interface may provide control over MRR spectrometer 200 (e.g., selection of desired ranges of frequencies for broadband and target measurements, timing intervals for sample injection). The processor 216 can Fourier transform the detected FID emissions to generate a corresponding MRR spectrum and display the MRR spectrum to a user via a graphical user interface.

[0047] The MRR spectrometer 200 generally measures the MRR spectrum of a sample by irradiating the sample with one or more excitation pulses of microwave and / or millimeter-wave radiation. The excitation pulses may be generated by one or more signal generators, such as an arbitrary waveform generator, a direct digital synthesizer, or a pulse pattern generator, and may be filtered, frequency multiplied, and / or upconverted with appropriate circuitry. The MRR spectrometer 200 may include a single signal generator shared by the broadband spectrometer 230 and the target spectrometer 220, or one signal generator for the broadband spectrometer 230 and another signal generator for the target spectrometer 220. If the MRR spectrometer 200 includes multiple signal generators, these signal generators may be locked to a common time / frequency standard to ensure they are synchronized and phase coherent. The excitation pulses generated by the signal generators may include both broadband and narrowband pulses generated simultaneously. The source antennas of the broadband spectrometer and the target spectrometer respectively emit broadband and narrowband excitation pulses to probe a sample in the vacuum chamber.

[0048] Components of a sample can be excited by one or more excitation pulses if the excitation pulses contain frequency components that match the characteristic rotational frequencies of the analyte. When the sample components are excited, they may emit FID signals for several microseconds. The receiver antennas of the broadband spectrometer and target spectrometer detect the FID signals (e.g., using heterodyne detection) emitted by the sample components in response to the broadband and narrowband pulses, respectively. Each FID signal may then be digitized by digitizer 214. In some instances, the FID signals may be mixed down to an intermediate frequency range so that they can be digitized by a narrower bandwidth digitizer, as discussed in more detail below with respect to Figures 3B and 3C. Processor 216 receives the time-domain digitized FID signals and performs a Fourier transform to generate an MRR spectrum.

[0049] The processor 216 may then use an MRR spectral library containing MRR spectra of previously characterized molecules to identify and / or quantify individual components of the analyte based on the MRR spectra. For example, data obtained from broadband and targeted measurements may be used to determine relative representations, such as the number density ratio or mass ratio of one chemical species to another in the sample. More generally, multiple MRR measurements may be used to determine various ratios of components in the sample, including, but not limited to, enantiomeric ratios, isomeric ratios, isotopologue ratios, and isotopomer ratios. Unassigned peaks in the MRR spectrum can be further analyzed for characterization using theoretical predictions of related species.

[0050] The processor 216 may record the FID signal with fine time resolution (e.g., at a sampling rate of 5-10 Hz) to allow the digitizer to achieve the desired throughput speed and the processor to perform data handling operations without dead time. The processor 216 may continuously measure and record the MRR spectrum of the sample as it is injected into the vacuum chamber. In some cases, the processor records and processes all MRR spectra. In other cases, the processor 216 records all of the time-domain data and Fourier transforms only the segments corresponding to the "interesting" output from the sampling inlet to conserve processing resources and reduce total processing time. The processor may discard unprocessed or uninterrogated time-domain and / or Fourier-domain data.

[0051] Further description of the various components of MRR spectrometer 200 is provided below. Further details regarding the operation of an MRR spectrometer and / or exemplary demonstrations of MRR spectroscopy may be found in U.S. Pre-Grant Publication No. 2022 / 0196582, entitled "Highly Selective Chromatography—Molecular Rotational Resonance Spectroscopy Systems and Methods," filed November 5, 2021, and incorporated herein by reference in its entirety.

[0052] 2.1 Broadband and Targeted Spectrometers The broadband spectrometer 230 of the MRR spectrometer 200 performs broadband measurements by generating broadband excitation pulses, irradiating a sample with the excitation pulses using a source antenna (e.g., a horn antenna), and receiving FID emissions by the sample in response to the excitation pulses using a receiver antenna (e.g., another horn antenna). The broadband spectrometer 230 may include a dedicated signal generator for generating the broadband excitation pulses. Alternatively, a single signal generator may be shared between the broadband spectrometer and the target spectrometer (see, for example, the MRR spectrometer 400 in FIG. 4A described below). For example, the signal generator may simultaneously generate both broadband and narrowband pulses and transmit the respective pulses to the source antennas of the target spectrometer and the broadband spectrometer accordingly.

[0053] In one non-limiting example, broadband spectrometer 230 can perform broadband measurements 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 an FID signal emitted by the sample in response to the chirped pulse. For details on chirped-pulse Fourier transform MRR spectroscopy, see, for example, the following U.S. patents: 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," each of which is incorporated herein by reference in its entirety. Wideband measurements can be performed across a segment of the entire band at once or across segments (e.g., a 2 GHz or 4 GHz segment of the 6-18 GHz band) to reduce data rates as disclosed in U.S. Pat. No. 8,873,043, entitled "Segmented Chirped-Pulse Fourier Transform Spectroscopy," each of which is incorporated herein by reference in its entirety.

[0054] The target spectrometer 220 of the MRR spectrometer 200 performs target measurements by generating narrowband excitation pulses, illuminating a sample with the excitation pulses via a source antenna, and receiving FID emissions by the sample in response to the excitation pulses via a receiver antenna. The target measurements may be facilitated, in part, by a Fabry-Perot resonator 222, which may be used to amplify excitation and / or FID emissions at desired frequencies and / or suppress undesired excitation and / or FID emissions at undesired frequencies. The target spectrometer 220 may include a dedicated signal generator for generating the narrowband excitation pulses. Alternatively, a single signal generator may be shared between the broadband spectrometer and the target spectrometer, as described above and below. The target spectrometer 220 can acquire, process, and store target measurement data relatively quickly, for example, at rates of 2 Hz, 3 Hz, 5 Hz, 10 Hz, or faster. This measurement speed can be increased by performing a direct Fourier transform on the field-programmable gate array (FPGA) that acquires the data.

[0055] Because the excitation frequency or frequencies and target species are known in advance, the target spectrometer 220 can operate without "species recognition" capabilities. Targeted MRR measurements typically involve looking at single line times versus intensity, since the measured intensity is proportional to the species concentration. If the target species and / or other information (e.g., separation parameters of a gas chromatography or liquid chromatography system) are known in advance, the target spectrometer 220 may be pre-programmed to target different spectral lines or bands for different species.

[0056] Broadband and targeted spectrometers may generally perform broadband and targeted measurements in one or more frequency bands in the microwave spectrum (e.g., across the 4-18 GHz band, including all values ​​and subranges therebetween) and / or the millimeter-wave spectrum (e.g., 75-110 GHz, 260-290 GHz, or 520-580 GHz, including all values ​​and subranges therebetween). The bandwidth of the targeted measurements performed by targeted spectrometers may generally be smaller than the bandwidth of the broadband measurements performed by broadband spectrometers. For example, the bandwidth of the targeted measurements may be 1 MHz or less, including all values ​​and subranges therebetween. The bandwidth of the broadband measurements may be greater than 5 MHz.

[0057] MRR spectrometer 200 may perform broadband and targeted measurements at non-overlapping frequencies. For example, broadband spectrometer 230 may perform measurements from 8 to 18 GHz, while targeted spectrometer 220 may perform measurements at 6 GHz.

[0058] (2.2 Vacuum Chamber) Vacuum chamber 202 may generally be designed so that multiple spectrometers (e.g., broadband spectrometer and target spectrometer of MRR spectrometer 200) can interact with the same volume of sample injected into the chamber. When multiple MRR measurements (e.g., broadband and target measurements) are performed simultaneously, multiple pulses (e.g., broadband and narrowband pulses) and FID emissions in response to a pulse may be simultaneously present in vacuum chamber 202, which may distort the measurements. In some instances, to reduce or mitigate interference between the respective broadband and target measurements, components of broadband spectrometer 230 and narrowband spectrometer 220 may be arranged to reduce the amplitude of electromagnetic radiation associated with broadband spectrometer 230 that reaches the detector of target spectrometer 220, or vice versa.

[0059] When both broadband and targeted MRR measurements are performed simultaneously, cavity ring modes (e.g., resonant modes determined by the shape and dimensions of the vacuum chamber cavity) may also be excited, for example, due to the broad frequency range of the broadband excitation pulse. To reduce the effect of the cavity ring on the measurements, RF absorbing material may be added to the inner walls of the vacuum chamber 202 to absorb at least a portion of the cavity ring modes. The quality factor of the vacuum chamber 202 may also be designed to be sufficiently low so that any cavity ring modes excited within approximately 5 microseconds dissipate. The FID signal generated in response to the broadband pulse may also be time-gated to reduce detection of cavity ring modes, since cavity ring modes typically dissipate faster than FID emission. Any artifacts in the detected FID signal caused by the cavity ring may also be digitally removed from the MRR spectrum by measuring a background spectrum in the absence of a sample.

[0060] 2.3 Sampling Inlets and Sample Sources The sampling inlet 206 may generally be fluidly coupled to a sample source (not shown) that provides a sample of the analyte analyzed by the MRR spectrometer 200. The sampling inlet 206 may inject the sample into the vacuum chamber in several ways, including, but not limited to, a continuous flow based on a trigger, pulsed injection at predetermined time intervals, and / or pulsed injection. Additionally, the sample source of the MRR spectrometer 200 may include various components for preparing the sample for measurement, including, but not limited to, a gas chromatograph, a liquid chromatograph, a programmable temperature vaporizer, and / or the like. The MRR spectrometers disclosed herein may generally include one or more sampling inlets 206 fluidly connected to the same or different sources. For example, some of the MRR spectrometers described below include two sampling inlets, one for injecting the analyte into the vacuum chamber 202 and the other for injecting a standard sample into the vacuum chamber 202.

[0061] FIG. 3 shows a sampling inlet 306 that can be used in the MRR spectrometer of FIG. 2 to inject both analyte and standard samples through a shared pulsed solenoid valve 368. This sampling inlet 306 has a three-port sample delivery design, including an analyte sample port 362 for receiving the analyte, a standard sample port 364 for receiving the standard sample, and a purge port for purging the shared sample volume when switching between the standard and analyte samples. This design allows for a high degree of sample introduction matching for the standard and analyte samples, since the same pulsed nozzle source 368 is used to inject both into the vacuum chamber 202. Because the sampling inlet 306 provides identical sample injection characteristics for the standard and analyte, it is particularly useful in applications where high measurement accuracy is desirable, such as stable isotope analysis.

[0062] An alternative sampling interface may include separate solenoid injectors for the reference and analyte to eliminate sample crossover contamination. The two sample injection systems may be positioned symmetrically within or around the vacuum chamber 202 so that pulse-jet expansion occurs primarily in the same active volume of the vacuum chamber 202. Alternatively, two separate sample injection systems, connected to a gas source using flexible tubing, may be placed on a movable mount that allows the nozzle of each sample injection system to be translated to the same injection position. It is also possible to use two separate nozzles embedded in one of the cavity mirrors using a coaxially oriented beam resonator design.

[0063] In one non-limiting example, the sample source of the MRR spectrometer 200 can include or be connected to a gas chromatograph (GC, not shown) with a temperature-regulated flow interface. The GC has a carrier gas source that flows a carrier gas, such as helium, hydrogen, neon, or argon, through a column. The carrier gas pushes analytes, which may have many different chemical constituents including isomers, isotopes, isotopes, and isotopologues, through the column and into a vacuum chamber via the flow interface. The analytes can be siphoned, accumulated, volatilized, and injected into the column (periodically) from a continuous stream or streams of gas or liquid as needed, so that the gas chromatograph effectively samples the continuous stream, much like an analog-to-digital converter (ADC) samples an analog signal.

[0064] Some or all of the analyte components may propagate through the column at different rates and therefore appear at the end of the column at different times. If these times are separated widely enough, the components can be resolved at the output of the column. Other components, e.g., isomers, may co-elute, i.e., appear at the output of the column at or near the same time, and therefore cannot be resolved using GC alone.

[0065] The carrier gas pushes the (at least partially separated) components through an interface into the vacuum chamber so that the MRR spectrometer 200 can measure the MRR spectra of the components. This interface allows for sample injection through a GC column or directly into the vacuum chamber (e.g., for pure compounds or simple mixtures that do not require GC separation). 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 MRR spectrometer (rather than through a GC), while other samples can be injected through a GC.

[0066] In another non-limiting example, the sample source of the MRR spectrometer 200 includes or is connected to a liquid chromatograph (LC). In this example, the sample source can include a volatilization interface for volatilizing at least a portion of the analyte for injection into the vacuum chamber. For details about volatilization interfaces, see U.S. Pregrant Publication No. 2021 / 0302340 A1, which is incorporated herein by reference in its entirety.

[0067] The sampling interface 206 may also be fluidly coupled to a second carrier gas source. The second carrier gas source flows a second carrier gas through the interface to push or propel the analyte components into the vacuum chamber. 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.

[0068] The sampling interface 206 may be coupled (in fluid communication) with a (chiral) tag source. By mixing chiral tags from the chiral tag source with the analyte components in the interfacial reservoir, the chiral tags bind to different components. The chiral tags alter the moment of inertia of different enantiomers between the analyte components, allowing the enantiomers to be resolved and quantified from their MRR spectra, as described below. The tag source may store and supply other types of tags, including polar molecules for tagging nonpolar molecules that do not have MRR spectra, to produce complexes that have a dipole moment and can therefore be detected by MRR.

[0069] The sample source may include an auxiliary (universal) detector, such as a thermal conductivity detector (TCD), to trigger the MRR measurement based on the output of the GC column. This auxiliary detector may be either in-line (sampling the same gas stream) or split (such as an FID or MS detector where the analyte is destroyed). When the auxiliary detector detects a peak in the GC output, it sends a trigger signal to the processor, which in turn triggers the emission of excitation pulses from the broadband and target spectrometers and measures and analyzes the resulting FID signal.

[0070] The MRR spectrometers disclosed herein can also be connected to sample sources that do not include a GC or LC. For example, the sample source can include a programmable temperature vaporizer for vaporizing the analyte and then injecting the analyte into the vacuum chamber of the MRR spectrometer 200.

[0071] The processor 216 of the MRR spectrometer 200 may also record all MRR data, as described above, and discard MRR data that does not map to a chromatographic peak sensed by the auxiliary detector. Additionally, auxiliary detector data may be combined with the MRR data to provide a more complete analysis of the analyte; for example, the auxiliary detector may sense components without dipole moments, while isotopic information from the MRR spectrometer 200 may complete the characterization of other components. For details on triggering MRR spectroscopy, 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.

[0072] The MRR spectrometer 200 may also be configured to analyze samples with high molecular weight analytes. In a typical MRR spectroscopy system with a flow cell operating at room temperature or above, the upper molecular weight limit may be approximately 150 amu, and sensitivity at molecular weights between 100 and 150 amu may be limited because MRR spectra of room-temperature molecules tend to be much weaker below 150 amu. However, using a pulse-jet ultrasonic expansion source, continuous-wave jet, or buffer-gas cooling cell can rotationally cool molecules for MRR analysis while keeping them in the gas phase. Therefore, in some examples, the sampling inlet 206 of the MRR spectrometer 200 may include a pulse-jet expansion source, continuous-wave jet, or buffer-gas cooling cell to analyze molecules with higher molecular weights (e.g., up to 400 amu or greater) via this rotational cooling.

[0073] In one non-limiting example, a pulse-jet supersonic nozzle can be used to inject analytes into the measurement chamber of the MRR spectrometer 200. The nozzle can accept one or more gas streams, including, but not limited to, an inlet for the analyte component flow, a vent valve inlet for rapid removal of solvent or other volatile matrix components, and an optional inlet for an (additional) carrier gas. The inlets can be made of 1 / 16-inch (1.5875 millimeter) tubing and heated to at least 300°C to accept samples at high temperatures (e.g., the direct inlet of a GC column). These tubing can be formed of PEEK plastic, although connections can alternatively be made of metal(s) with better thermal properties. A gas connection (e.g., a 1 / 4-inch (6.35 millimeter) gas connection) for the carrier gas is optionally provided, and a purge gas can be used to rapidly clean the sample. Multiple gas streams can be combined in a reservoir, which can have a volume of about 500 mL or less.

[0074] The combined gas stream can be co-expanded with the rare carrier gas through a pinhole nozzle approximately 1 mm in diameter. The pinhole nozzle is rapidly opened and closed by a solenoid valve sealed with a Teflon poppet. With each gas pulse (approximately 1 ms duration), the analyte component travels through the pinhole into the MRR spectrometer measurement chamber, which is placed under high vacuum (approximately 10 -6 The pressure is maintained at 1000 kPa (1 Torr). During the passage through the pinhole, the molecules undergo many collisions with the carrier gas. These collisions reduce the rotational temperature of the analyte components by approximately 1-2 K.

[0075] In one non-limiting example, analyte components separated by a GC column can be injected into a vacuum chamber 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 (psig) (about +0.01379 to about +0.03447 megapascals). The nozzle pulses at 10 Hz. With each pulse injection, the nozzle injects about 1 nmol of analyte component and 1 μmol of neon into the vacuum chamber. This corresponds to about 10 to 15 mL / min (STP) of carrier gas passing through the MRR spectrometer 200. This is comparable to the typical flow rate of a wide-bore GC column. Injecting neon carrier gas into the nozzle can enhance pulsed valve operation. When neon carrier gas is injected into the nozzle, the neon will dominate the rotational cooling caused by the ultrasonic pulse injection of the analyte components into the vacuum chamber, so the GC column can be operated with a different carrier gas, such as hydrogen or helium. Both carrier gases can be injected into the vacuum chamber. The pulse-jet supersonic nozzle can be used with GC columns or with other sample source components, including but not limited to LC and programmable temperature vaporizers.

[0076] (2.4 Timing Controller) The MRR spectrometers disclosed herein can include a timing controller (e.g., timing circuitry and / or electronics) for synchronizing the operation of multiple spectrometers (e.g., broadband spectrometers and target spectrometers) and / or sampling inlets (e.g., via pulsed valve drivers). For example, the timing controller may synchronize the emission of broadband excitation pulses and the emission of resonant excitation signals generated by signal generators associated with the broadband spectrometers and target spectrometers. The timing controller can further synchronize receivers to receive the respective FID signals emitted in response to the broadband and resonant excitation pulses and / or the operation of a processor (e.g., to process the digitized FID signals and determine the ratio of analytes).

[0077] In one non-limiting example, the timing controller may include a frequency standard (e.g., a rubidium atomic clock) that provides a reference clock signal (e.g., a 10 MHz clock signal) for synchronizing the operation of various components within the MRR spectrometer. For example, the broadband spectrometer and the target spectrometer may each include a dedicated signal generator for generating broadband and narrowband pulses, and a detector (e.g., a receiver antenna) for detecting emissions from the sample. The signal generator, processor, and electronics for triggering the pulsed valve driver may be operably connected to the frequency standard and locked to the same reference clock signal. In another non-limiting example, the timing controller may include a pulse generator and / or pulse pattern generator to generate timing signals for each spectrometer and / or sampling inlet of the system.

[0078] If the system includes a single signal generator that generates both the broadband excitation pulse and the target excitation pulse, that signal generator can also be used to trigger the pulsed valve driver or to emit the broadband excitation pulse and the target excitation pulse in response to a pulse trigger from another circuit or component that controls the pulsed valve driver. This signal generator can also emit one or more local oscillator signals for heterodyne detection of the FID pulses emitted by the sample in response to the broadband and target excitation pulses. If the broadband and target excitation pulses and the local oscillator signals are all generated by the same signal generator, they should be phase coherent with each other, regardless of whether the signal generators are locked to separate frequency references.

[0079] In another embodiment, the system may include separate signal generators for the target and broadband measurements. In this case, both signal generators may be locked to the same frequency reference, with one signal generator triggering the other signal generator and other electronics, or both signal generators responding to triggering pulses from another circuit or component locked to the same frequency reference. In this case, each signal generator emits a local oscillator (LO) signal for heterodyne detection of the corresponding FID pulse.

[0080] In yet another embodiment, the system may include a separate generator for generating each LO signal in parallel with the signal generator that generates the excitation pulses, which may be, for example, circuitry integrated into the broadband spectrometer or target spectrometer.

[0081] (2.5 Heterodyne Signal Detection and Digitization) Figures 4A and 4B illustrate analog front ends 440a and 440b, respectively, for detecting and digitizing the FID pulses emitted by a sample in response to broadband and target excitation pulses. The analog front end 440a in Figure 4B has separate channels for the broadband and target FID pulses. Each channel includes a mixer 442a, 442b for mixing the corresponding FID pulse with a corresponding local oscillator (LO) signal to generate an intermediate frequency (IF) output at a lower center frequency (e.g., 100 MHz vs. 2-18 GHz for the FID pulse). Each channel also includes its own digitizer (ADC) 444a, 444b for digitizing the corresponding IF output and providing the resulting digitized FID pulse to a corresponding input channel of the processor 216, which Fourier transforms the digitized FID pulse to generate the broadband and target MRR spectra of the sample. Each ADC 444a, 444b can have a bandwidth of up to approximately 3 GHz. The FID pulses can have a bandwidth of up to about 2 GHz.

[0082] The analog front-end 440b shown in FIG. 4B also includes separate mixers 442a, 442b for the FID pulses emitted from the sample in response to the broadband excitation pulse and the target excitation pulse. However, in this case, the FID signals are mixed with LO signals at different frequencies f1 and f2 into different, non-overlapping IF bands (e.g., 75-125 MHz and 150-200 MHz). More typically, the IFs may range from approximately 0 GHz to 3 GHz, including all values ​​and subranges therebetween. The LO frequencies may range from approximately 2 GHz to approximately 18 GHz, depending on the FID center frequency and target IF band. The bandwidth of each frequency channel may be approximately 30 MHz. The spacing between the different IF ranges may range from approximately 500 MHz to approximately 1500 MHz. These frequency-downconverted FID signals emitted by mixers 442a, 442b are digitized by the same digitizer 446, which provides digitized outputs to processor 216 for spectrum generation and other additional processing. In other words, the mixers 442a, 442b and the LO operate to frequency multiplex the wideband and target FID signals onto separate frequency multiplexed channels within the same ADC 446 band, allowing the ADC 446 to digitize both signals simultaneously. The processor 216 can separate or demultiplex the signals by filtering the signals in the frequency domain. Spurs in the signals can also be filtered during post-processing using background measurements.

[0083] 4A and 4B are non-limiting examples. In yet another embodiment, the broadband and target FID pulses may be combined before mixing and / or amplification. In this example, the analog front end includes a single mixer and a single ADC.

[0084] 4A and 4B may also be used in an MRR spectrometer that includes two target spectrometers, where the FID pulses generated for each target spectrometer are mixed and / or digitized in separate channels.

[0085] (3.MRR spectrometer cavity) Figures 5A-5H illustrate different cavity designs for MRR ratiometers. Each cavity design features two spectrometers: one target spectrometer and another either a target spectrometer or a broadband spectrometer. Both spectrometers are positioned to excite and detect FID signals from the same or overlapping volumes of samples (e.g., analyte sample or standard sample) within the vacuum chamber at the same or overlapping times.

[0086] 5A shows an exemplary MRR ratiometer 500a having a vacuum chamber 502 shaped as a hexagonal intersection with a first axis 521 for a target spectrometer 520, a second axis 531 for a broadband spectrometer 530, and a third axis (not shown) for other hardware (e.g., ports for connecting to a vacuum pump and / or pressure gauge). The first, second, and third axes may be orthogonal to one another. For example, the third axis may point into and out of the page of FIG. 5A.

[0087] Target spectrometer 520 is a Balle-Flygare spectrometer having a Fabry-Perot cavity formed by a fixed mirror 522a and a movable mirror 522b at opposite ends of vacuum cavity 502 along a first axis 521. A motorized translation stage 524 can move movable mirror 522b back and forth along first axis 521 through a travel range L to change the resonant frequency of the target spectrometer. Target spectrometer 520 also includes a source antenna 526a and a receiver antenna 526b mounted coaxially with fixed mirror 522a. Broadband spectrometer 530 includes a source horn antenna 532a and a receiver horn antenna 532b within vacuum chamber 502 at opposite ends of a second axis 531.

[0088] MRR ratiometer 500a also includes hardware control circuitry 510, timing control circuitry 512, analog front end and digitizer 514, processor 516, and one or more signal generators 518. These electronics function as described above: signal generator 518 generates target and broadband excitation pulses; analog front end and digitizer 514 amplifies, filters, and digitizes the FID signal; and processor 516 calculates the Fourier transform of the FID signal and the ratio of species of interest. Hardware control circuitry 510 and timing control circuitry 512 synchronize the emission of the excitation pulses with the injection of analyte or standard samples into vacuum chamber 502 using a sampling interface or nozzle 506 fluidly coupled to a sample source 508.

[0089] During operation, source antenna 526a emits a target excitation pulse, and receiver antenna 526b receives an FID signal from the sample in response to the target excitation pulse. Similarly, source horn antenna 532a emits a broadband excitation pulse, and receiver horn antenna 532b detects an FID signal from the sample in response to the broadband excitation pulse. Generally, FID emissions travel in the same direction as the corresponding excitation pulse. Therefore, FID emissions stimulated by a broadband pulse are more likely to propagate along second axis 531 and less likely to propagate along first axis 521. Similarly, FID emissions associated with narrowband pulses are less likely to enter axis 531 of broadband spectrometer 530. Furthermore, the frequency ranges of detection for each spectrometer 520, 530 are typically different. Therefore, even if a portion of the FID emission associated with a broadband (narrowband) pulse travels along an axis associated with the target (broadband) spectrometer 520 (530), the detector of that spectrometer should not detect that FID emission.

[0090] Figures 5B and 5C illustrate MRR ratiometers 500b and 500c, respectively, each containing two target cavity-enhanced FTMW (Balle-Flygare) spectrometers 520a, 520b with orthogonal cavity axis orientations. Standard and analyte samples are introduced into the spectrometer region 501 where the cavity volumes of the two FTMW spectrometers 520a, 520b overlap. As shown in Figure 5B, this can be achieved by positioning pulsed nozzle sources 506b for the analyte and standard samples above (and / or below) the plane of the FTMW spectrometers 520a, 520b. Alternatively, the standard and analyte samples can be introduced using nozzles 506, 506' positioned at 45° diagonals, as shown in Figure 5C.

[0091] Figure 5D shows an MRR ratiometer 500d with a pair of coaxial, overlapping, polarization-multiplexed microwave cavities within a vacuum chamber. The first cavity is defined by a fixed curved mirror 582 and a movable flat mirror 584 on a first translation stage 585. The second cavity is defined by a fixed curved mirror 582 and a movable wire-grid polarizer 586 between the fixed curved mirror 582 and the movable flat mirror 584. The movable wire-grid polarizer 586 is disposed on a second translation stage 587 that is actuable independently of the first translation stage 585 and may be mounted to the first translation stage 585 as shown in Figure 5D. Two nozzles 560, 562 inject the analyte and standard sample, respectively, into a common sample region between the fixed curved mirror 582 and the movable wire-grid polarizer 586. Source and receiver antennas (not shown) can be attached to or through the fixed curved mirror 582 or the movable flat mirror 584. This is similar to source antenna 526a and receiver antenna 526b in Figure 5A, although attachment to fixed curved mirror 582 tends to simplify the design and construction of MRR ratiometer 500d.

[0092] The wire grid polarizer 526 transmits radiation polarized orthogonal to the grid wires and reflects radiation polarized parallel to the grid wires. As a result, radiation orthogonal to the grid wires resonates in the first cavity, and radiation parallel to the grid wires resonates in the second cavity. The first cavity has a resonant frequency defined by the distance between the fixed curved mirror 582 and the movable flat mirror 584, and the second cavity has a resonant frequency defined by the distance between the fixed curved mirror 582 and the movable wire grid polarizer 526. Both cavities are semi-confocal because they have a movable flat reflective surface and a (shared) curved mirror.

[0093] The MRR spectrometer 500d shown in Figure 5D has at least two advantages over the cross-cavity design shown in Figures 5A-5C: (1) its small footprint makes it more suitable for analytical chemistry laboratory space, and (2) the cylindrical symmetry of the cavity axes for both molecular species makes it easier to arrange dual pulse-jet sample sources to inject samples into the same active volume of the spectrometer.

[0094] Wire grid polarizers with excellent performance are commercially available. For example, Millitech, Inc. produces wire grid polarizers for microwave and THz applications that can be fabricated up to 9 inches (228.6 mm) in diameter. Microwave optics uses 0.001-inch (0.0254 mm) diameter tungsten wires. A wire grid polarizer with 200 wires per inch can have over 99% transmission for radiation at 200 GHz when the electric field is polarized perpendicular to the wires and less than 1% transmission when the electric field is parallel to the wire windings. Performance at lower frequencies used in MRR spectroscopy (e.g., below 20 GHz) can be even better. Wire grid polarizers are also excellent reflectors at THz frequencies. Future manufacturing advances may make it possible to fabricate high-performance wire grid polarizers with spherical geometries for use as confocal cavity mirrors.

[0095] Figures 5E-5H show MRR ratiometers 500e-500h, respectively, with a folded cavity using a wire grid polarizer 540 as a polarizing beam splitter. In each of these MRR ratiometers 500e-500h, the wire grid polarizer 540 is fixed at a 45° angle relative to the axis of the microwave cavity of the Balle-Flygare (target) spectrometer. Again, the source and receiver antennas (not shown) can be mounted with or through any of the mirrors of these ratiometers, but mounting them within the fixed mirrors tends to simplify design and construction.

[0096] 5E and 5F, the folded cavity is a confocal cavity defined by fixed curved mirrors 542, 542′ on one side of wire grid polarizer 540 and a pair of movable curved mirrors 544 on the other side of wire grid polarizer 540. In FIGS. 5G and 5F, the folded cavity is a semi-confocal cavity defined by either a fixed flat mirror 546 and a movable curved mirror 544, or by a fixed curved mirror 542 and a movable flat mirror 544′.

[0097] In Figures 5E, 5G, and 5H, analyte nozzle 560 injects the analyte sample into the vacuum chamber from the top, and standard nozzle 562 injects the standard sample into the vacuum chamber from the side. In Figure 5F, analyte and standard nozzles 560, 562 inject samples into the vacuum chamber through fixed curved mirror 542'. In all of these MRR ratiometers 500e-500h, the nozzles inject the analyte and standard samples into substantially identical or overlapping volumes within the vacuum chamber for more consistent FID stimulation and detection. Both MRR ratiometers 500e and 500f include source and receiver antennas (not shown) attached to or passing through fixed curved mirror 542, 542', similar to source antenna 526a and receiver antenna 526b in Figure 5A.

[0098] In all of these MRR ratiometers 500e-500h, the wire grid polarizer 540 acts as a polarizing beamsplitter, transmitting microwave radiation orthogonal to its wires and reflecting microwave radiation polarized parallel to its wires. The transmitted radiation reflects off one movable mirror 544, 544' and returns through the wire grid polarizer 540 to the receiver antenna and fixed curved mirrors 542, 542'. The reflected radiation reflects off another movable mirror 544, 544' back to the wire grid polarizer 540, which reflects the radiation back to the receiver antenna and fixed mirrors 542, 542', 546.

[0099] In these MRR ratiometers 500e-500h, the movable mirrors 544, 544′ can be moved independently of each other to tune the cavity resonances of the orthogonal polarizations to different molecular resonance frequencies. Having separate movable mirrors to define the cavity lengths of the two orthogonal electric field polarizations provides additional space for positioning antennas to couple microwave pulses into and out of the cavity for FTMW measurements.

[0100] Some of the MRR ratiometer designs described herein can be extended to measure two different ratios simultaneously. For example, as shown in Figure 5A, an MRR ratiometer with a dual-polarized horn antenna can simultaneously monitor two different "strong" signals using two orthogonal polarizations. Incorporating a wire-grid polarizer into the spectrometer cavity allows for the simultaneous measurement of two separate "weak" signals (from sample impurities). This MRR ratiometer can simultaneously perform two different ratiometric measurements, provided the pulse generation and receiver systems are expanded to allow for additional excitation pulses and coherent FID signal measurements. Similarly, if two of the cavity instruments incorporating wire-grid polarizers are arranged in a crossed configuration, the resulting MRR ratiometer can monitor four separate frequencies or perform two independent ratio measurements for each sample injection.

[0101] (4. MRR Ratio Measurement Method) 6A and 6B show an exemplary method for simultaneously performing broadband and target measurements to determine the ratio of one component of a sample to another component of the sample (e.g., isomer, isotopologue, and / or isotopomer ratio, enantiomeric ratio). The steps of this method may be performed using a timing controller to synchronize the injection of analyte into the vacuum chamber, the broadband measurement, and the target measurement. For example, the steps of this method may be performed according to the frequency standard described above.

[0102] As shown in FIG. 6A, MRR ratio measurements involve injecting a sample (either an analyte or a standard) into the vacuum chamber of an MRR ratiometer (602). Once the sample is in the chamber, the MRR ratiometer measures a first MRR spectrum of the sample with a first excitation pulse (604) and simultaneously measures a second MRR spectrum of the sample with a second excitation pulse (606). Both excitation pulses may be resonant pulses with different resonant frequencies, or one excitation pulse may be a broadband (e.g., chirped) pulse and the other a resonant pulse. The MRR ratiometer quantifies the first and second components (chemical species) of the sample based on the first and second MRR spectra, respectively (608, 610), compares the amounts of the first and second components (612), and determines the ratio of the first and second components (e.g., isomer, isotopologue, and / or isotopomer ratios, enantiomeric ratios) (614).

[0103] Generally, the period during which a sample pulse or portion of the sample stream can be exposed to the broadband may be significantly longer than the FID dephasing time. For example, as shown in Figure 6B, the sample pulse may reside in the vacuum chamber for a period on the order of milliseconds, while the FID emission may occur in a period on the order of microseconds. In some applications, the MRR ratiometer may use multiple broadband and / or narrowband pulses to probe a particular sample pulse or portion of the sample stream and detect the corresponding FID emission in response to the broadband and / or narrowband pulses (e.g., Figure 6B). The duration of the spectrometer's excitation pulses is analyte-dependent and may be the same or different. Ideally, detection is strictly simultaneous (although this is not a strict requirement, pulses may be alternated to reduce or avoid crosstalk).

[0104] For example, an MRR spectrometer may irradiate a sample pulse multiple times with the same broadband and / or narrowband pulse to improve the signal-to-noise ratio of the detected FID signal (e.g., by summing spectra obtained from multiple broadband or narrowband pulses). In another example, an MRR spectrometer may perform a frequency sweep on the same sample pulse in which multiple narrowband pulses covering a set of frequencies are generated in sequence, and record FID signals in response to the narrowband pulses at different frequencies.

[0105] The MRR ratiometers disclosed herein are capable of performing simultaneous broadband and target measurements on the same sample, but can also perform separate broadband and target measurements on separate samples, if desired.

[0106] (5. Use Case Examples) Molecular rotational resonance (MRR) spectroscopy, or simple rotational spectroscopy, is a high-resolution spectroscopic technique with high structural specificity. This technique measures the spectroscopic transfer of rotational kinetic energy between quantized energy levels. The parameters A, B, and C in the Hamiltonian operator for the kinetic energy of rotation around the molecular center of mass of a rigid molecule, known as rotational constants, are inversely proportional to the moment of inertia calculated in the principal axis system. As a result, any difference in the mass distribution of molecular structures generates differences in rotational constants and distinguishable spectra. This allows MRR spectroscopy to distinguish between molecular isomers, including isotopic isomers, providing unique analytical chemistry capabilities. Advances in quantum chemistry have made it possible to estimate molecular rotational constants with high precision using optimized (equilibrium) geometries. Therefore, reliable identification of distinct chemical species is possible through comparison of experimental and theoretical rotational constants. Finally, spectrometers for MRR spectroscopy using pulse-jet expansion of analytes seeded in a vacuum in a noble gas (commonly neon) have spectral resolution unparalleled in the field of analytical chemistry. This feature of the instrument for MRR spectroscopy allows for the analysis of individual chemical species within a complex sample matrix without prior chemical separation by chromatographic methods.

[0107] MRR spectroscopy is particularly useful for making measurements that are difficult or impossible to perform using established analytical chemistry techniques such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (often coupled with gas chromatography), infrared spectroscopy, and electron spectroscopy. Two emerging application areas for MRR spectroscopy are chiral analysis and stable isotope analysis. The MRR ratiometer described herein provides unique measurement capabilities for these two areas. The MRR ratiometer of the present invention is also of general use for accurately determining the relative abundance of pairs of chemically distinct species.

[0108] (5.1 Chiral Analysis) Chirality is a subtle aspect of molecular structure. A molecule is chiral if it has a non-superimposable mirror image. These "handed" forms of a molecule are called enantiomers. In isolation, enantiomers have identical chemical and physical properties.

[0109] However, when placed in a homochiral or handheld environment, enantiomers may exhibit different chemical behavior. One application where chirality can be effective is in medicinal chemistry, where the homochiral environment of the body can result in different safety and efficacy profiles for the two enantiomers of a drug. As a result, current guidance from the Food and Drug Administration is that active pharmaceutical ingredients (APIs) should be manufactured as single enantiomers. The challenge of enantiomer-specific drug production drives developments in both synthetic chemistry and chemometrics. Furthermore, new approaches to API production that employ continuous manufacturing concepts using flow chemistry methods may also involve the use of analytical chemistry techniques. In particular, for these manufacturing techniques, it is desirable to have high-speed chiral analysis at high enantiopurity limits, which are difficult to achieve by chiral chromatographic analytical methods. The MRR ratiometer of the present invention can perform spectroscopic chiral analysis with high accuracy and speed.

[0110] Chiral analysis using the MRR ratiometer of the present invention is built on chiral tag rotational spectroscopy. Chiral tag measurements provide accurate determination of the enantiomeric excess (EE) of an analyte. EE is a measure of the relative abundance of two non-superimposable mirror-image stereoisomers. This measurement approach uses chiral derivatization to generate spectroscopically distinguishable species from enantiomers (if the enantiomers have identical rotational spectra without derivatization). In chiral tag rotational spectroscopy, derivatization is achieved using non-covalent attachment of small chiral molecules, or tags, via cluster formation in pulse-jet expansion of a mixture of analyte and tag molecules. Unlike chiral derivatization approaches in NMR spectroscopy, no additional chemical synthesis steps are performed. Furthermore, in this approach, derivatization does not affect the stereochemistry of the chiral center (a process known as racemization, which compromises analytical accuracy). A small number of small, volatile chiral molecules have been shown to efficiently complex with a wide range of chemical analytes, enabling accurate enantiomeric excess determination of these compounds within mixtures. For more details on chiral tagging, see, for example, U.S. Patent No. 11,237,104, entitled "Cavity-Enhanced Fourier transform Spectroscopy for Chiral Analysis," which is incorporated herein by reference in its entirety. Further details regarding the use of the MRR spectrometers described herein for chiral analysis can also be found in the Appendix.

[0111] Figures 7A and 7B illustrate 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 7A. Weakly bound complexes stabilized by a combination of hydrogen bonding, van der Waals, and other forces can be efficiently formed in a pulsed ultrasonic expansion nozzle. The resulting diastereomeric complexes have well-defined moments of inertia and can be resolved by the capabilities of MRR spectroscopy, as described above and shown in Figure 7B. Chiral analysis is important in a wide range of applications, including pharmaceuticals, environmental analysis, and metabolite analysis.

[0112] More specifically, Figure 7A shows that the enantiomers of verbenone (an example of a chiral molecule) have the same moments of inertia and MRR spectra. However, when a chiral tag (in this case, (S)-3-butyn-2-ol) is complexed with the two enantiomers, a difference in the moments of inertia occurs, which produces two species with different moments of inertia and therefore distinct MRR spectra. The 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).

[0113] Figure 7B shows one spectral line each for the heterochiral and homochiral complexes shown in Figure 7A. In a racemic mixture of butynol tags, the two complexes are seen at approximately equal intensities (these vary depending on the complex dipole moment, formation ratio, and other factors). With pure (S)-3-butyn-2-ol as the tag, the lines are seen at different intensities. These line ratios may be used to calculate the enantiomeric ratio and / or enantiomeric excess of the butynol in a sample.

[0114] Chiral monitoring may be incorporated into the MRR ratiometer of the present invention as an auxiliary system for introducing gas-phase chiral tags. For example, the MRR ratiometer of the present invention may include an additional valve connected to a source that supplies gas-phase chiral tags at the interface of the gas chromatography outlet before the sampling inlet (e.g., nozzle). The chiral tags may be mixed with the sample eluting post-column (e.g., after the sample exits the gas chromatography outlet). Furthermore, a spectral library containing known compounds and their respective MRR spectra may include compounds with chiral tags. For the MRR ratiometer 200 of Figure 2, broadband and targeted measurements may be used to determine the enantiomeric ratio between two enantiomers in a sample, the enantiomeric excess (e.g., purity of a chiral material), the absolute configuration of a chiral component in a sample, and / or the achiral purity of the sample.

[0115] 5.2 Chiral Tag Enantiomeric Excess (EE) Analysis as a Ratio Determination Quantitative measurement of EE is possible using intensity information in the rotational spectra of weakly bound complexes formed during pulse-jet expansion of chiral analytes with small chiral molecules (chiral tags, or simply tags) used to distinguish between analyte enantiomers. Homochiral complexes are defined as 1:1 clusters of analyte and tag molecules in which both species have the same chiral designation. Here, the designation is defined by the sign of optical rotation (+ or -). Heterochiral complexes are formed by clusters of opposite chirality. The geometries of homochiral and heterochiral complexes have different mass distributions (and therefore different rotational constants) and generate distinct MRR spectra. The derivation of the equation used to determine EE has one assumption: the number densities of homochiral and heterochiral complexes are linearly proportional to the number densities of the tag and analyte in the pulse-jet expansion. As a result, the intensities of transitions in homochiral and heterochiral MRR spectra can be described.

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[0116] Chiral tag measurements use transition intensities from two different samples. In one case, a racemic sample of tag is used (this is the standard sample). In the second measurement, a high enantiopure tag sample is used (EE is known from a separate analysis). EE determination uses normalized transition intensities, defined using the intensities of the spectra using the racemic and enantiopure tag samples, as follows:

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[0117] Using the normalized intensities, the ratio R is

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[0118] An example of the precision and accuracy of chiral tag rotational spectroscopy is illustrated in the EE determination of 1,1,1-trifluoro-propan-2-ol (trifluoroisopropanol, TFIP). This analysis is a special case of chiral tag spectroscopy called autotagging. In this case, the analyte serves as both tag and analyte, and the analysis uses homochiral and heterochiral dimers of TFIP formed by pulse-jet expansion. In this case, the EE is determined from:

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[0119] Figure 8 shows the measured calibration curves, with at least three replicate measurements for each sample. Standard samples of known EE were prepared from a mixture of an enantiopure sample of TFIP (EE = 99%) and a racemic TFIP sample. The amounts of enantiopure and racemic samples used in each mixture were determined gravimetrically. The EE of each standard sample was measured using the "auto-tag" variant of chiral tag rotational spectroscopy on a broadband chiral pulsed FTMW spectrometer, using the TFIP dimer for analysis. EE determinations for each standard sample were performed in triplicate. Chiral tag rotational spectroscopy provides the EE directly from the measurement, eliminating the need for analyte samples of known EE. The characteristics of this measurement can be summarized as follows: (1) the measured EE is exactly the standard sample EE (i.e., there is no need to calibrate the measurement using samples with known EE from a different chiral analytical technique), (2) the method is accurate across the entire EE range, and (3) the measurements have high precision, as indicated by the small spread in replicate measurements of each standard sample.

[0120] The chiral tag method can be implemented in an MRR ratiometer. As can be seen in equation (5), the EE determination is based on the accurate determination of the quantity R, which is defined using equations (3) and (4).

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[0121] Using the formulation of Equation (8), the MRR ratiometer of the present invention (1) uses a cavity-enhanced FTMW spectrometer to measure weak signals in enantiopure tag measurements, shortening measurement times; (2) simultaneously measures both homochiral and heterochiral signals in each sample injection; and (3) allows two different samples to be injected into the instrument: one in which enantiopure tags are used, and one in which the reference measurement uses a racemic tag sample.

[0122] Overall, the precision and accuracy of chiral tag rotational spectroscopy exceeds the "gold standard" method of chiral gas chromatography. However, chiral tag rotational spectroscopy has the potential for high-speed operation. The MRR ratiometer described herein makes it possible to realize the potential of the technology.

[0123] (5.3 Current EE Measurement Limits) Fast EE measurements were demonstrated using a cavity-enhanced Fourier transform microwave (FTMW) spectrometer based on a Balle-Flygare instrument. With existing instrument designs, FTMW spectrometers can only measure at a single resonant frequency. For chiral tag measurements, this means that the signal levels from the homochiral and heterochiral complexes are measured separately by retuning the resonator when switching between the two species. A significant limitation for the analysis of high-enantiopurity samples (EE ~100) is the presence of low-abundance chiral tagged complexes at low levels. As a result, significant signal averaging is typically required to measure the rotational spectroscopy signal associated with this chiral tagged complex down to the limit of quantitation (typically a signal-to-noise ratio of 10:1).

[0124] During the course of a measurement, the signal is subject to several sources of drift: time-dependent changes in sample composition, temperature changes that modify the amplifier gain of the microwave electronics, and position drift of the cavity mirrors that modify the cavity resonance position relative to the molecular transition frequency. As a result, accurate EE determination is difficult at high EE limits, i.e., the limits of importance in chemical manufacturing. The MRR ratiometer instrument described herein has two key design additions that address signal drift: simultaneous measurement of the two signals used to determine EE (obtained from the rotational transitions of chemically distinct homochiral and heterochiral tag complexes), and the ability to rapidly switch between the sample being measured (analyte) and the standard sample used for measurement calibration.

[0125] (5.4 Stable isotope analysis) A second analytical chemistry application enabled by the MRR ratiometer of the present invention is site-specific stable isotope analysis. Stable isotope analysis is an established field of analytical chemistry that measures small variations in the relative abundance of an isotope relative to a reference standard. For example, common analysis can measure the relative abundance of a chemical relative to a reference standard. 13 C: 12 Determine variations in C isotope ratios. These variations are a small fraction of the average natural abundance of the stable isotopes. 13 For C stable isotope analysis, changes are reported at the parts per thousand level.

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[0126] Currently, stable isotope analysis is most commonly performed using isotope ratio mass spectrometry, in which the analyte is converted to CO2 via combustion and the resulting CO2 is 13 C: 12 The C ratios are measured relative to a standard CO2 sample. A limitation of this approach is that it does not capture all of the position-specific information about the isotopic variations, i.e., the δ ratios of each of the different carbon atoms in the molecular structure. 13 The limitation is that the C measurement is lost. 13 The C isotopic species have the same mass and are therefore acceptable for mass spectrometry-based detection. 13 Isotopes that are isomeric with a C nucleus but with different structural positions for isotopic substitution are not easily distinguished by mass spectrometry.

[0127] 13 Unlocking the complete chemical information in C stable isotope measurements requires a technique that can provide a distinct, fully resolved signature for each isotope and detect signals with a signal-to-noise ratio of 100,000:1 (or higher), so that changes from natural abundance can be measured with sufficient precision. With the exception of very small molecules, for which infrared spectroscopy is applicable, only two analytical chemistry techniques have the potential to meet these measurement criteria: NMR spectroscopy and MRR spectroscopy. 13 Attempts to develop robust analytical methods using C NMR spectroscopy have been reported. 13 C: 12It has been shown that there is significant site-specific variation in C isotope ratios (e.g., using vanillin). However, ensuring the instrumental portability of the results presents challenges. Additionally, NMR spectroscopy is a slow measurement method requiring expensive, large equipment with significant maintenance requirements.

[0128] The MRR ratiometer disclosed herein has the potential to open the field of site-specific stable isotope analysis to a wide range of analytes. MRR spectroscopy can achieve the desired 100,000:1 signal-to-noise ratio requirement for many molecules. Furthermore, each isotopomer has a distinct mass distribution and therefore a distinct rotational spectrum. Finally, the high spectral resolution of the MRR spectrometer ensures that these distinct spectral signatures can be measured without spectral overlap. A restatement of equation (9) shows that stable isotope measurements can be performed with an MRR ratiometer, where the two signals being monitored are: 12 C and singly substituted 13 for one of the C isotopes.

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[0129] Furthermore, measurements should be performed on two different samples (analyte and standard). For an MRR ratiometer to be successful, measurement accuracy must be primarily, or in some cases, weaker. 13 It is preferable to remove the effect of number density variations in the amount of sample injected in each measurement cycle, as limited only by the signal-to-noise ratio of the C isotopomer MRR transitions.

[0130] 5.5 Characterization of signal fluctuations in pulsed jet MRR measurements The fluctuations of individual rotational transitions in MRR spectroscopy have been characterized by measurements on a broadband chirped-pulse FTMW spectrometer. This measurement represents an ideal case: (1) in broadband measurements, all transitions are detected with the same valve pulse, so that signal fluctuations caused by the sample injection system are apparent; (2) all transitions are measured using a single excitation pulse, so that pulse amplitude fluctuations do not affect measurement characterization; and (3) all transitions are detected by the same receiver system, so that there are no variations from different detector components. Broadband MRR spectrometers offer excellent performance for ratio measurements. However, measurements are impractical. For applications such as EE determination of high-enantiopurity samples and stable isotope analysis using low-abundance stable isotopes, many spectral averages are often required to reduce the noise level to a point where weaker impurity species can be detected with sufficient sensitivity for accurate ratio determination. Both the long measurement time and high sample consumption for broadband measurements prevent this approach from being a useful analytical chemistry method. The MRR ratiometer described herein includes a cavity-enhanced FTMW detection system for measuring lower abundance impurities such that measurement time and sample consumption are significantly reduced.

[0131] Figures 9A-9C show signal characterization measurements using TFIP. This sample is also used to validate ratiometric measurements for EE determination and stable isotope measurements, as described below. Pulse-jet expansion of TFIP generates clusters (used for chiral tag EE determination) so that both monomer and dimer signals are observed. The measurement dataset is 10 back-to-back spectral acquisitions with 40,000 averages of the broadband free induction decay signal.

[0132] Figure 9A shows the signal fluctuations of individual MRR spectroscopic transitions observed in measurements of TFIP. The signal fluctuations or variations in consecutive measurements are shown in Figure 9B for the TFIP dimer transition and Figure 9C for the TFIP monomer transition. In both cases, the intensity fluctuations, measured as the standard deviation of 10 separate measurements, are proportional to the intensity of the transition. Furthermore, the proportionality constant is twice as large for the dimer transition compared to the monomer transition. These results indicate that the intensity fluctuations originate from variations in the number density of sample injection cycles.

[0133] Figures 9A-9C show that there is significant variation in signal levels caused by variations in sample volume in each pulse-jet injection cycle. The source of variation is determined by examining the standard deviation of the signal over 10 measurements for both the monomer and dimer signals. The signal level is proportional to the number density, or concentration, of the species in the pulse-jet expansion. If the signal variation is caused by variations in number density, the variation (measured by the standard deviation of the signal over 10 trials) should be proportional to the average signal. Furthermore, because the number density of dimers is proportional to the square of the number density of monomers, the percent variation of the dimer signal should be twice that of the monomer signal. The expectation of signal variation caused by variations in number density of the sample effect is met, as shown in Figures 9A-9C.

[0134] As can be seen from Figure 9A, if the signal fluctuations are caused by pulse-to-pulse fluctuations in number density from the pulse valve used to inject the gas mixture into the vacuum chamber, the signal levels for all transitions are correlated. This behavior allows for the design of a ratiometer. Simultaneous measurements of the signal intensities for two transitions and determining the ratio for each measurement eliminates the effect of number density fluctuations. The effectiveness of the running ratio measurement for each signal acquisition cycle can be evaluated by considering the ratio fluctuations. Consider the signal intensities I1 and I2 used to measure the ratio R.

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[0135] TFIP's 13 C: 12 Consider the example of measuring the ratio variation of the C signals. The results in panel C of Figure 2 show that for independent measurements of two isotope signals, for one different sample injection cycle, the fractional variation of the isotope ratio is expected to be determined by the pulse-to-pulse variation of the number density.

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[0136] The results of Equation (13) and Equation (15) have different behavior. For example, in Equation (13), the observed specific 13 The same variation will be observed regardless of the signal strength (and therefore signal-to-noise ratio) of the C isotopomer transition. Conversely, if ratio measurements remove the effects of pulse-to-pulse number density variations in sample injection from the ratio determination,13 A dependence on the C transition strength is observed in the variation of the isotope ratio in sequential measurements.

[0137] Figure 10 shows the results from a broadband spectrometer. 13 C: 12 C isotope ratio measurement results are shown. These results show that specific gravity measurements using simultaneous intensity measurements for the same sample injection cycle are consistent with removing the effect of number density fluctuations on the ratio determination. There are three MRR spectroscopic transitions used in the ratio determination. The x-axis represents the 12 C transition intensity ( 13 C transition intensity). TFIP has three carbon atoms, so these 12 For each C transition, three measurable 13 C: 12 C ratios are shown as individual data points. The variation of the ratio is given on the y-axis. The isotope ratios are measured for three different numbers of signal averages.

[0138] The solid line in Figure 10 is calculated from equation (15) and the measured noise characteristics of the chirped pulse FTMW spectrometer used to make the measurements. The fluctuations in the isotope ratio are calculated by the ratio 13 The ratiometric approach depends on both the signal averaging number and intensity of the C transitions. These are characteristics of ratiometric measurements that eliminate the influence of number density fluctuations associated with the sample injection system. In the specific case of stable isotope analysis, the measurement accuracy of the isotope ratio is limited only by the noise level of the spectrometer detection system, which can be reduced by additional signal averaging. These results show that for ideal measurements, the ratiometric approach mitigates the measurement limitations caused by the pulsed jet source used to inject the sample into the spectrometer. Below, we discuss the effects of weak transitions (e.g., 13 The ratiometer designs described in this disclosure with enhanced measurement sensitivity to C transition intensities are shown to maintain the improved measurement characteristics shown here for the ideal case.

[0139] 6. Experimental Ratio and Enantiomeric Excess Determination 11-14 show the results for the molecular sample TFIP prepared using an MRR ratiometer such as that shown in FIG. 5A. 13 Experimental ratio measurements of C isotopes and enantiomeric excess determination are shown. As mentioned above, the MRR ratiometer design in Figure 5A is an extension of the Balle-Flygare cavity-enhanced FTMW spectrometer and uses two horn antennas to perform broadband MRR measurements across the FTMW spectrometer cavity. The horn antennas do not require any mechanical motion control to tune the measurement system to resonance. Furthermore, because the horn antennas have no resonance frequency limitations, multiple transition intensities can be monitored in each measurement cycle. This capability increases the flexibility of analytical chemistry measurement methods.

[0140] The MRR ratiometer in Figure 5A is particularly useful when impurity species are present at low abundance, which is a key measurement challenge in analytical chemistry. Because one species is present at a much higher abundance, the signal can be detected with good sensitivity without the enhancement provided by a resonant-cavity FTMW spectrometer. The pulse-jet source for injecting the sample into the vacuum chamber is located on the cavity axis and the horn antenna axis ( 13 The beam can be either perpendicular to the C (used for the C measurement) or placed within the cavity mirror of a coaxially oriented beam resonator arrangement.

[0141] 11 to 14 show the EE determination and 13 We demonstrate the performance of this MRR ratiometer for both C stable isotope ratio measurements. TFIP was used as the test sample. 13 C stable isotope ratio studies were performed on the monomer spectra. EE determination of high enantiopure samples of (R)-TFIP was performed using the transition ratio of TFIP dimers using an auto-tagging method. Two different prototypes were constructed: a ratiometer in which the sample was injected into a shared active sample volume and used a nozzle positioned above the plane of the instrument and centered on the common active volume; 13 It was used for C stable isotope test measurements. The COBRA configuration, in which the sample is injected coaxially with the cavity-enhanced measurement system, was used for EE determination.

[0142] Figures 11 to 13 show 13 Figure 11 shows the results of a C stable isotope measurement. Figure 11 shows that the ratiometer design of Figure 5A eliminates the effect of continuous number density fluctuations in pulse-jet sample introduction. The black data points indicate 12 C (normal species) and 13 Measurements of C isotopomer transition intensities are shown for 10 consecutive 10 kAvg measurements. 12 C and 13 Both of the C transition intensity measurements have measurement variations measured by the standard deviation of 10 different intensity measurements of approximately 10%. This results in a constant percentage of signal variations consistent with the number density variations that are responsible for the signal variations discussed above. If the measurements were uncorrelated, these variations would be 1 3 C:1 2 This results in a 14% variation in the C stable isotope signal ratio. However, the data in Figure 11 are obtained by simultaneous measurements with the same pulse jet expansion. 12 C and 13 C indicates that the transition intensities are highly correlated (i.e., the data points lie on a line rather than the circular pattern observed for uncorrelated transition intensities). Ratio measurements are indicated by data points with lighter shading. The variation in the ratio is 0.8%, which is monitored within the instrument. 13 The performance is limited by the signal-to-noise ratio of the C transition. This performance shows that the effect of number density variations, which would have resulted in a 14% variation in the signal ratio, is mitigated by the design of the MRR ratiometer.

[0143] In addition to mitigating the effects of successive measurement variations in the number density of the sample injection system, a successful ratiometer should also reduce, or more preferably, eliminate, long-term drift in the signal ratio. In the design of the MRR ratiometer of the present invention, long-term drift is eliminated by using a nozzle or pair of nozzles that allow the analyte and standard to be injected separately into the vacuum chamber. Alternating between analyte and standard on a timescale faster than instrument drift eliminates these effects in measurements that observe differences between the characteristics of the analyte and standard (such as stable isotope measurements, as shown in Equation (9)).

[0144] Figure 12 shows the results of 60 back-to-back ratiometer measurements of TFIP. 13 C: 12 The stable isotope ratios of C are shown (1 kJ / min for each measurement, approximately 1.5 min per observation). A slow drift in the ratio is clearly observed. The effect of switching between standard and analyte samples and measuring the ratio difference in equation (9) can be simulated by treating all other measurements as "reference" uptakes. In this case, the analyte and standard samples are identical, so the average δ 13 C is expected to be zero and should not be flat. The results of this analysis are shown in Figure 13. The mean value of the modeling of the reference-analysis switching is zero within the measurement uncertainty: δ 13 C=3.7‰±10‰. δ 13 The uncertainty in C (σ=10‰) is weaker 13 This is consistent with the signal-to-noise ratio on the C transition, and consequently, deeper signal averaging is expected to lower measurement variability.

[0145] Test measurements for the EE determination of TFIP also demonstrated that the MRR ratiometer (Figure 5A) mitigates the effects of variations in pulsed sample injection number density, resulting in highly accurate measurements. Furthermore, the ability to introduce both the analyte and standard sample yields highly accurate EE determinations using the results of Equations (7) and (8). The (R)-TFIP used in the test measurements had an EE of 95.9, measured in separate analytical chemistry determinations. For EE determination applications, analyte measurement poses challenges due to the low abundance of the second enantiomer. As a result, cavity-enhanced detection is often used, and even then, significant signal averaging may be required. To achieve the desired measurement precision, the MRR ratiometer can eliminate the effects of variations in sample number density through simultaneous measurement of the signals of the homochiral (strong) and heterochiral (weak) dimer transitions.

[0146] For the three TFIP test measurements, five consecutive measurements were performed. As expected from the results shown in Figures 9A-9C, a certain percent variation was observed between the individual homochiral and heterochiral measurements, 7% for both the cavity-enhanced heterochiral dimer complex transition intensity and the homochiral transition intensity. If these signals were uncorrelated, then

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[0147] Figure 14 shows the EE measurement results for five consecutive measurements of (R)-TFIP using the MRR ratiometer in Figure 5A. For each measurement, four separate EE values ​​are reported. This is possible because chirped pulses were used to measure the stronger homochiral signal on the horn antenna detection axis, and four strong homochiral transitions were observed in each measurement cycle that can be used to calculate the EE.

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[0148] Further examples, embodiments, and / or modifications to the embodiments of the invention described herein are described by one or more of the following numbered paragraphs.

[0149] (Item 1) a first transmitter operatively connected to the at least one signal generator and configured to irradiate the sample with the first excitation pulse; a first receiver for receiving a first free induction decay (FID) signal emitted by the sample in response to the first excitation pulse; a second transmitter operatively connected to the at least one signal generator and configured to irradiate the sample with a second excitation pulse while the first transmitter irradiates the sample with the first excitation pulse; a second receiver for receiving a second FID signal emitted by the sample in response to the second excitation pulse; and a processor operatively connected to the first receiver and the second receiver for determining a relative representation of at least two components of the sample based on the first FID signal and the second FID signal.

[0150] (Item 2) Item 1. The MRR measurement apparatus according to item 1, wherein the first transmitter is configured to irradiate the sample with a first excitation pulse along a first axis, and the second transmitter is configured to irradiate the sample with a second excitation pulse along a second axis different from the first axis.

[0151] (Item 3) 2. The MRR measurement apparatus according to item 1, wherein the first excitation pulse is a first resonant excitation pulse and the second excitation pulse is a second resonant excitation pulse.

[0152] (Item 4) Item 10. The MRR measurement apparatus of item 1, wherein the first excitation pulse comprises a broadband excitation pulse and the second excitation pulse comprises a resonant excitation pulse encompassing a bandwidth narrower than the bandwidth of the broadband excitation pulse.

[0153] (Item 5) Item 5. The MRR measurement apparatus of item 4, wherein at least one signal generator is configured to generate a resonant excitation pulse at a frequency outside the band of the broadband excitation pulse.

[0154] (Item 6) 6. The MRR measurement apparatus according to item 5, wherein the resonant excitation pulse and the broadband excitation pulse include frequency components in the range of 2 GHz to 18 GHz.

[0155] (Item 7) 5. The MRR measurement apparatus according to item 4, wherein the broadband excitation pulse is a chirped excitation pulse.

[0156] (Item 8) 5. The MRR measurement apparatus of item 4, further comprising a pair of mirrors disposed within the vacuum chamber and forming a Fabry-Perot cavity having a resonance corresponding to the resonant excitation pulse.

[0157] (Item 9) 2. The MRR measurement apparatus according to item 1, wherein the first excitation pulse and the second excitation pulse include frequency components in the range of 2 GHz to 18 GHz.

[0158] (Item 10) 2. The MRR measurement apparatus of claim 1, wherein the first receiver is configured to mix the first FID signal into a first intermediate frequency range, and the second receiver is configured to mix the second FID signal into a second intermediate frequency range different from the first intermediate frequency range.

[0159] (Item 11) Item 11. The MRR measurement apparatus according to item 10, wherein the first intermediate frequency range and the second intermediate frequency range are from about 0 GHz to about 3 GHz.

[0160] (Item 12) Item 10. The MRR measurement apparatus according to item 1, further comprising a timing controller operably connected to the sampling interface and the at least one signal generator to trigger the emission of the first excitation pulse and the second excitation pulse in coordination with the injection of the sample.

[0161] (Item 13) Item 13. The MRR measurement apparatus of item 12, wherein the timing controller includes a frequency standard configured to generate a reference frequency clock signal to synchronize the emission of the first excitation pulse with the emission of the second excitation pulse.

[0162] (Item 14) Item 13. The MRR measurement apparatus of item 12, wherein the timing controller is further configured to trigger the first receiver to receive the first FID signal, the second receiver is further configured to receive the second FID signal, and the relative indication is a ratio of the first component to the second component.

[0163] (Item 15) Item 1. The MRR measurement apparatus according to item 1, further comprising a sampling interface in fluid communication with the vacuum chamber, for injecting a sample into the vacuum chamber.

[0164] (Item 16) Item 1. The MRR measurement device described in item 1, further comprising at least one analog-to-digital converter operably connecting the first receiver and the second receiver to a processor to convert analog outputs of the first receiver and the second receiver into digital signals for the processor.

[0165] (Item 17) Item 17. The MRR measurement device according to item 16, wherein at least one analog-to-digital converter has a bandwidth in the range of 0 GHz to 3 GHz.

[0166] (Item 18) 2. The MRR measurement apparatus of claim 1, wherein the first excitation pulse is in a first polarization state and the second excitation pulse is in a second polarization state different from the first polarization state, and further comprising a wire grid polarizer disposed in the vacuum chamber to transmit the first excitation pulse and reflect the second excitation pulse.

[0167] (Item 19) Item 19. The MRR measurement apparatus of item 18, wherein the wire grid polarizer is positioned to reflect the second excitation pulse toward the second transmitter.

[0168] (Item 20) Item 19. The MRR measurement apparatus of item 18, wherein the wire grid polarizer is positioned to reflect the second excitation pulse at an angle relative to the second transmitter.

[0169] (Item 21) Item 10. The MRR measurement apparatus according to item 1, wherein the relative representation of the at least two components is one of an enantiomeric ratio, a ratio of two isotopic species, or a ratio of two different chemical species.

[0170] (Item 22) 1. A method for measuring a ratio of a first component of a sample to a second component of the sample, the method comprising: injecting the sample into a vacuum chamber; obtaining a first molecular rotational resonance (MRR) spectrum of the sample; simultaneously obtaining the first MRR spectrum of the sample, obtaining a second MRR spectrum of the sample using a second excitation pulse; and determining the ratio of the first component to the second component based on the first MRR spectrum and the second MRR spectrum.

[0171] (Item 23) 23. The method of claim 22, wherein measuring the first MRR spectrum comprises emitting a first excitation pulse in a first direction, and measuring the second MRR spectrum comprises emitting a second excitation pulse in a second direction different from the first direction.

[0172] (Item 24) 23. The method of claim 22, further comprising quantifying isomer, isotopologue, and / or isotopomer ratios between components of the sample based at least in part on the first MRR spectrum and the second MRR spectrum.

[0173] (Item 25) 23. The method of claim 22, further comprising quantifying a first component of the sample based at least in part on the first MRR spectrum, quantifying a second component of the sample based at least in part on the second MRR spectrum, and comparing the amount of the first component to the amount of the second component.

[0174] (Item 26) Item 23. The method of item 22, wherein the sample contains about 10 to about 100 compounds, measuring the first MRR spectrum comprises measuring free induction decay (FID) signals of about 10 to about 100 compounds in the sample, and measuring the second MRR spectrum comprises measuring at least one FID signal from a single compound in the sample.

[0175] (Item 27) 23. The method of claim 22, further comprising identifying at least one unknown component of the sample based at least in part on the second MRR spectrum.

[0176] (Item 28) 23. The method of claim 22, wherein the first excitation pulse is a first resonant excitation pulse and the second excitation pulse is a second resonant excitation pulse emitted at a different frequency than the first resonant excitation pulse.

[0177] (Item 29) 23. The method of claim 22, wherein the first excitation pulse is a broadband excitation pulse and the second excitation pulse is a resonant excitation pulse.

[0178] (Item 30) 30. The method of claim 29, further comprising measuring a response of the vacuum chamber to the broadband excitation pulse, wherein measuring the second MRR spectrum includes taking into account the response of the vacuum chamber to the broadband excitation pulse.

[0179] (Item 31) 23. The method of claim 22, further comprising attaching a chiral tag to at least one component in the sample prior to measuring the first MRR spectrum and the second MRR spectrum, identifying an enantiomer in the sample based on one of the first MRR spectrum or the second MRR spectrum, and / or determining the enantiomeric excess of the component of the sample based on the first MRR spectrum and the second MRR spectrum.

[0180] (7. Conclusion) All parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on the particular application in which the teachings of the present invention are used. The foregoing embodiments are presented primarily by way of example, and it should be understood that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein.

[0181] Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of each element of the exemplary implementations without departing from the scope of the present disclosure. The use of numerical ranges does not exclude equivalents outside the range that fulfill the same function to produce the same results.

[0182] Also, various inventive concepts may be embodied as one or more methods, at least one example of which is provided. Acts performed as part of a method may, in some instances, be ordered differently. Thus, in some inventive implementations, the acts of a given method may be performed in an order different from that specifically illustrated, which may include performing some acts simultaneously (even when such acts are shown as sequential acts in an exemplary embodiment).

[0183] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0184] All definitions defined and used herein should be understood to control for dictionary definitions, definitions in documents incorporated herein by reference, and / or ordinary meanings of the defined terms.

[0185] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0186] As used herein in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some instances and disjunctively present in other instances. 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, whether related or unrelated to those elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B" can refer in one embodiment to A only (optionally including elements other than B), in another embodiment to B only (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0187] As used herein in the specification and 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 interpreted as being inclusive, i.e., including at least one of a number or list of elements, but including two or more, and optionally, additional unlisted items. When used in the claims, it refers to the inclusion of exactly one element of a number or list of elements. Generally, when used herein, the term "or" should only be interpreted as indicating exclusive alternatives, such as "one or the other, but not both," or "only one" or "exactly one." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0188] As used herein in the specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, synonymously, "at least one of A or B," or, synonymously, "at least one of A and / or B") can refer in one embodiment to the presence of at least one, optionally more than one, A, where B is absent (optionally including elements other than B); in another embodiment to the presence of at least one, optionally more than one, B; in yet another embodiment to the presence of at least one, optionally more than one, A, and at least one B (optionally including other elements); etc.

[0189] In the claims and the foregoing specification, all transitional phrases, such as "comprising," "carrying," "having," "containing," "involving," "holding," "comprising," and the like, are to be understood to be open-ended, i.e., including, but not limited to: Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. a vacuum chamber for holding the sample; at least one signal generator for emitting a first excitation pulse and a second excitation pulse; a first transmitter operatively connected to the at least one signal generator to illuminate the sample with the first excitation pulse; a first receiver for receiving a first free induction decay (FID) signal emitted by the sample in response to the first excitation pulse; a second transmitter operatively connected to the at least one signal generator, the second transmitter irradiating the sample with the second excitation pulse while the first transmitter irradiates the sample with the first excitation pulse; a second receiver for receiving a second FID signal emitted by the sample in response to the second excitation pulse; a processor operatively connected to the first receiver and the second receiver for determining a relative representation of at least two components of the sample based on the first FID signal and the second FID signal; A molecular rotational resonance (MRR) measurement device comprising:

2. 2. The MRR measurement apparatus of claim 1, wherein the first transmitter is configured to irradiate the sample with the first excitation pulse along a first axis, and the second transmitter is configured to irradiate the sample with the second excitation pulse along a second axis different from the first axis.

3. 2. The MRR measurement apparatus of claim 1, wherein the first excitation pulse is a first resonant excitation pulse and the second excitation pulse is a second resonant excitation pulse.

4. 2. The MRR measurement apparatus of claim 1, wherein the first excitation pulse comprises a broadband excitation pulse and the second excitation pulse comprises a resonant excitation pulse encompassing a bandwidth narrower than the bandwidth of the broadband excitation pulse.

5. 5. The MRR measurement apparatus of claim 4, wherein the at least one signal generator is configured to generate the resonant excitation pulse at a frequency outside the band of the broadband excitation pulse.

6. 6. The MRR measurement apparatus of claim 5, wherein the resonant excitation pulse and the broadband excitation pulse include frequency components in the range of 2 GHz to 18 GHz.

7. 5. The MRR measurement apparatus of claim 4, wherein the broadband excitation pulse is a chirped excitation pulse.

8. 5. The MRR measurement apparatus of claim 4, further comprising a pair of mirrors disposed within the vacuum chamber to form a Fabry-Perot cavity having a resonance corresponding to the resonant excitation pulse.

9. 2. The MRR measurement apparatus according to claim 1, wherein the first excitation pulse and the second excitation pulse include frequency components in a range of 2 GHz to 18 GHz.

10. 2. The MRR measurement apparatus of claim 1, wherein the first receiver is configured to mix the first FID signal into a first intermediate frequency range and the second receiver is configured to mix the second FID signal into a second intermediate frequency range different from the first intermediate frequency range.

11. 11. The MRR measurement apparatus of claim 10, wherein the first intermediate frequency range and the second intermediate frequency range are from 0 GHz to 3 GHz.

12. 2. The MRR measurement apparatus of claim 1, further comprising a timing controller operatively connected to the sampling interface and the at least one signal generator to trigger emission of the first excitation pulse and the second excitation pulse in coordination with injection of the sample.

13. 13. The MRR measurement apparatus of claim 12, wherein the timing controller includes a frequency standard configured to generate a reference frequency clock signal for synchronizing the emission of the first excitation pulse with the emission of the second excitation pulse.

14. 13. The MRR measurement apparatus of claim 12, wherein the timing controller is further configured to trigger the first receiver to receive the first FID signal, and the second receiver is further configured to receive the second FID signal, and the relative indication is a ratio of the first component to the second component.

15. The MRR measurement apparatus of claim 1 , further comprising a sampling interface in fluid communication with the vacuum chamber for injecting the sample into the vacuum chamber.

16. 2. The MRR measurement device of claim 1, further comprising at least one analog-to-digital converter operatively connecting the first receiver and the second receiver to the processor to convert analog outputs of the first receiver and the second receiver into digital signals for the processor.

17. 17. The MRR measurement apparatus of claim 16, wherein the at least one analog-to-digital converter has a bandwidth in the range of 0 GHz to 3 GHz.

18. the first excitation pulse is in a first polarization state and the second excitation pulse is in a second polarization state different from the first polarization state; 10. The MRR measurement apparatus of claim 1, further comprising a wire grid polarizer disposed within the vacuum chamber, the wire grid polarizer transmitting the first excitation pulse and reflecting the second excitation pulse.

19. 20. The MRR measurement apparatus of claim 18, wherein the wire grid polarizer is positioned to reflect the second excitation pulse toward the second transmitter.

20. 20. The MRR measurement apparatus of claim 18, wherein the wire grid polarizer is positioned to reflect the second excitation pulse at an angle relative to the second transmitter.

21. 10. The MRR measurement apparatus of claim 1, wherein the relative representation of the at least two components is one of an enantiomeric ratio, a ratio of two isotopic species, or a ratio of two different chemical species.

22. 1. A method for determining a ratio of a first component of a sample to a second component of said sample, comprising: Injecting the sample into a vacuum chamber; obtaining a first molecular rotational resonance (MRR) spectrum of the sample; acquiring a second MRR spectrum of the sample using a second excitation pulse simultaneously with obtaining the first MRR spectrum of the sample; determining a ratio of the first component to the second component based on the first MRR spectrum and the second MRR spectrum; The method comprising:

23. 23. The method of claim 22, wherein measuring the first MRR spectrum comprises emitting a first excitation pulse in a first direction, and measuring the second MRR spectrum comprises emitting a second excitation pulse in a second direction different from the first direction.

24. 23. The method of claim 22, further comprising quantifying isomer, isotopologue, and / or isotopomer ratios between components of the sample based at least in part on the first MRR spectrum and the second MRR spectrum.

25. quantitating the first component of the sample based at least in part on the first MRR spectrum; quantitating the second component of the sample based at least in part on the second MRR spectrum; and comparing the amount of the first component with the amount of the second component; 23. The method of claim 22, further comprising:

26. the sample comprises 10 to 100 compounds; measuring the first MRR spectrum includes measuring free induction decay (FID) signals of 10 to 100 compounds in the sample; 23. The method of claim 22, wherein measuring the second MRR spectrum comprises measuring at least one FID signal from a single compound in the sample.

27. 23. The method of claim 22, further comprising identifying at least one unknown component of the sample based at least in part on the second MRR spectrum.

28. 23. The method of claim 22, wherein the first excitation pulse is a first resonant excitation pulse and the second excitation pulse is a second resonant excitation pulse emitted at a different frequency than the first resonant excitation pulse.

29. 23. The method of claim 22, wherein the first excitation pulse is a broadband excitation pulse and the second excitation pulse is a resonant excitation pulse.

30. measuring a response of the vacuum chamber to the broadband excitation pulse; 30. The method of claim 29, wherein measuring the second MRR spectrum includes taking into account the response of the vacuum chamber to the broadband excitation pulse.

31. attaching a chiral tag to at least one component in the sample prior to measuring the first MRR spectrum and the second MRR spectrum; identifying the enantiomers in the sample based on one of the first MRR spectrum or the second MRR spectrum, and / or determining the enantiomeric excess of the components of the sample based on the first MRR spectrum and the second MRR spectrum.

23. The method of claim 22, further comprising:

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