Method and apparatus for vibrational circular dichroism analysis of analytes - Patents.com

Abstract

An apparatus (1) for vibrational circular dichroism analysis of an analyte, comprising a laser source (2) for providing a laser beam (3), a beam splitter (5) for splitting the laser beam (3) into a sample light (6a) and a reference light (6b), a modulator (11) for modulating the polarization of the sample light (6a), a sample cell (14a) for interaction of the sample light (6a) with a sample containing an analyte, a reference cell (14b) for interaction of the reference light (6b) with a reference, a sample detector (15a) for detecting the sample light (6a) to obtain a sample light signal corresponding to the intensity of the sample light (6a), a reference detector (15b) for detecting the reference light (6b) to obtain a reference signal corresponding to the intensity of the reference light (6b), and a subtractor (16) for forming a difference signal between the sample signal and the reference signal. Also a corresponding method.

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for vibrational circular dichroism analysis of an analyte. [Background technology]

[0002] Circular Dichroism (CD) spectroscopy refers to the difference in absorbance of left and right circularly polarized light by a sample. It can therefore be used to study stereochemically related molecules in solution. A subfield of this research field is Vibrational Circular Dichroism (VCD), which deals with vibrational transitions, particularly in the mid-infrared region (MIR). This region is rich in bands characteristic of organic molecular bonds, allowing a wide range of applications of vibrational circular dichroism for stereochemical and biological studies. In the infrared region, the observed absorbance difference is typically on the order of 10 -4 ~10 -6 In addition, many solvents have strong absorption in the region of interest, reducing the throughput of the spectra, necessitating the use of very short optical path lengths in traditional mid-infrared (MIR) absorption spectroscopy. These properties are a significant drawback, as weak signals must be detected in the presence of relatively high noise levels. Most commercially available instruments based on Fourier transform infrared spectroscopy (FT-IR) rely on the averaging of repeated scans to achieve an acceptable signal-to-noise ratio in VCD measurements and allow proper interpretation of the spectra, which often results in measurement times of several hours per sample.

[0003] This shortcoming has motivated continuous improvements in interferometer design and in VCD devices in general, resulting in lower noise levels and fewer artifacts. In the case of VCD devices, careful optical design and polarization modulation schemes have made it possible to suppress the artifacts (see Nafie, LA Dual Polarization Modulation: A Real-Time, Spectral-Multiplex Separation of Circular Dichroism from Linear Birefringence Spectral Intensities. Appl. Spectrosc. 2000, 54 (11), 1634-1645).

[0004] At the same time, mid-infrared (Mid-IR) spectroscopy in general has seen significant improvements in terms of sensitivity due to the use of novel tunable laser sources. In particular, the advent of high-brightness external cavity quantum cascade lasers has allowed the use of longer optical path lengths, resulting in higher sensitivity and lower detection limits (see Akhgar, CK; Ramer, G.; Zbik, M.; Trajnerowicz, A.; Pawluczyk, J.; Schwaighofer, A.; Lendl, B. The Next Generation of IR Spectroscopy: EC-QCL-Based Mid-IR Transmission Spectros-copy of Proteins with Balanced Detection. Anal. Chem. 2020, 92 (14), 9901-9907). Combining quantum cascade lasers (QCLs) with VCD instruments was a logical step. S. Ludeke, M. Pfeifer and P. Fischer, J. Am. Chem. Soc., 2011, 133, 5704-5707 compare a custom-made QCL-based instrument with a commercially available FTIR-VCD instrument and demonstrate that QCLs are suitable for VCD.

[0005] Mid-infrared lasers such as QCLs provide very bright and polarized radiation, allowing for longer optical path lengths, even in strongly absorbing solvents like water, resulting in higher sample absorbance and easier sample handling. However, mid-infrared lasers such as QCLs introduce additional noise into the system that is not present in traditional thermal sources. Noise in laser sources can take the form of pulse-to-pulse fluctuations, intensity drift, spectral mismatch due to imperfect grating movements, and low-frequency noise, which adversely affect VCD results. Thus, the reduction in measurement time is not fully achieved compared to FTIR systems where the noise level does not change.

[0006] Furthermore, M. Bonmarin and J. Helbing, "A picosecond time-resolved vibrational circular dichroism spectrometer," Opt. Lett. 33, 2086-2088 (2008) show a VCD system in which an infrared laser pulse is split by a beam splitter. One beam is modulated by a photoelastic modulator and illuminated through a sample. Both beams are detected. However, this method does not allow accurate cancellation of noise, since the reference beam does not pass through the sample. Thus, absorption effects due to solvents and polarization-dependent beam splitting artifacts are not corrected. Furthermore, only every 50th period of the photoelastic modulator is used. Summary of the Invention

[0007] It is an object of the present invention to reduce or overcome one or more of the disadvantages of the prior art. In particular, it is an object of the present invention to provide a VCD method and apparatus that provides reduced noise and / or improved time resolution (i.e., more measurements per unit time).

[0008] The object of the present invention is to a) generating a laser beam, preferably at a wavelength in the infrared range; b) splitting the laser beam into a sample beam and a reference beam; c) modulating the polarization of the sample light; and d) interacting the sample light with a sample containing the analyte; e) interacting the reference light with a reference; f) measuring the intensity of the sample light to obtain a sample signal; g) measuring the intensity of the reference light to obtain a reference signal; h) subtracting the sample signal from the reference signal to obtain a difference signal.

[0009] Further, the object of the present invention is to 1. An apparatus for vibrational circular dichroism analysis of an analyte, comprising: a laser source for providing a laser beam; a beam splitter for splitting the laser beam into a sample beam and a reference beam; a modulator for modulating the polarization of the sample light; a sample cell for interaction of the sample light with a sample containing the analyte; a reference cell for interaction of said reference light with a reference; a sample detector for detecting the sample light to obtain a sample light signal corresponding to an intensity of the sample light; a reference detector for detecting said reference light to obtain a reference signal corresponding to the intensity of said reference light; a subtractor for forming a difference signal between said sample signal and said reference signal; This is achieved by an apparatus comprising:

[0010] By implementing a laser source in combination with a balanced detection scheme (splitting the laser light into sample and reference light and measuring the difference between them), the fluctuations can be compensated for by measuring the reference light and subtracting the sample and reference light signals (i.e., sample and reference signals), allowing us to take advantage of the high brightness of the laser source without introducing additional noise into the system. This allows for low-noise measurements with significantly improved time resolution, potentially opening up new applications of VCD spectroscopy, especially in biopharmaceutical applications.

[0011] The laser source preferably comprises a semiconductor laser (particularly tunable). The laser source preferably comprises a quantum cascade laser (QCL), particularly an external cavity QCL. Optionally, the laser source comprises an interband cascade laser (ICL). The laser beam is preferably pulsed. The sample detector and the reference detector each preferably comprise a HgCdTe detector (i.e., an MCT detector). Optionally, the apparatus comprises a balance detector, the balance detector comprising a sample detector, a reference detector, and a subtractor. Optionally, the balance module has a first output for a reference signal, a second output for a sample signal, and a third output for a difference signal (which may also be referred to as a balance signal). Optionally, the sample detector and / or the reference detector comprise a dark current compensation function.

[0012] The sample light is specifically modulated such that the sample light contains varying degrees of circular polarization. The sample light is specifically modulated to exhibit linear, elliptical, and / or circular polarization states. Optionally, the sample light is modulated according to a modulation period, such that the sample light has circular polarization at maxima and minima of the modulation period. The modulator modulates the light at a particular wavenumber (e.g., 1666.67 cm -1, i.e. 6 μm), particularly at the wavenumber of the laser beam, particularly at the maximum and minimum of the modulation period of the modulator. Optionally, the modulator is adjusted during the measurement (e.g., to achieve a quarter-wave phase difference at a number of different wavelengths, particularly at the maximum and minimum of the modulation period). Optionally, the sample comprises a solvent. Optionally, the reference comprises a solvent. Preferably, the sample and the reference comprise the same solvent. Optionally, both the sample and the reference comprise a solid sample (i.e., a pure solid material or a mixture of solids, e.g., pellets of KBr and analyte). Optionally, the sample and the reference comprise a gas (i.e., a pure gas or a mixture of gases, including the analyte in a matrix gas). Optionally, only the sample comprises the analyte (i.e., optionally, the reference does not comprise the analyte). The device may be contained in a housing and / or purged (constantly) with dry air to prevent interference from water vapor during the spectrum acquisition. In particular, the method and apparatus are for vibrational circular dichroism spectroscopy of analytes.

[0013] The infrared region is defined as a wavelength between 0.75 μm and 1000 μm. Optionally, in step a), the laser beam is generated at a wavelength in the mid-infrared region, defined as a wavelength between 2.5 μm and 50 μm. The beam splitter provides a transmitted light and a reflected light, one of which is used as a sample light and the other of which is used as a reference light. The beam splitter is preferably a 50 / 50 beam splitter. The sample light is in particular irradiated onto a sample cell, more particularly through a modulator and a sample cell at a sample detector. The reference light is in particular irradiated onto a reference cell, more particularly through a reference cell at a reference detector. The sample cell and / or the reference cell are preferably temperature stabilized. The subtractor may be an analog subtractor.

[0014] Preferably, the method further comprises the step of i) recovering the polarization dependent intensity change introduced by said analyte from said difference signal.

[0015] Optionally, the method further comprises the step of c1) modulating the polarization of said reference light (6b). Optionally, step c1) is performed substantially simultaneously with step c).

[0016] Optionally, the polarization dependent intensity change is due to a difference in absorption of left-handed and right-handed circularly polarized light in the sample or the analyte.

[0017] Optionally, the intensity signal is calculated from the difference signal and the reference signal and / or the sample signal.

[0018] Optionally, the method comprises scaling said polarization dependent intensity variation using said intensity signal.

[0019] Steps a) to h), preferably steps a) to i) (or a) to j) below), and further steps described herein, may be performed in any order and / or multiple steps may be performed simultaneously. Preferably, step c) is performed before step d).

[0020] Optionally, the laser beam is generated with pulses having a laser repetition frequency (i.e., laser pulse frequency). The sample light is modulated by a modulator, which imparts a change in polarization that oscillates periodically according to the modulator frequency. Optionally, the laser repetition frequency is equal to or greater than the modulator frequency. The change in polarization (i.e., optical retardation) that the modulator imparts to light passing through the modulator oscillates with the modulator frequency, where each period of the oscillating change in polarization (i.e., imparted phase difference) includes a maximum and a minimum (i.e., a maximum of negative phase difference). Optionally, the maximum and minimum values ​​include left-handed and right-handed circular polarization, respectively. That is, the sample light is modulated by a modulator, which operates with modulation periods that impart a periodic oscillating change in polarization (i.e., optical retardation), where each modulation period includes a maximum and a minimum, and at least one pulse of the laser beam is generated per modulation period. The laser repetition frequency is the frequency of pulse generation by the laser light source. Optionally, the laser repetition frequency is at least twice the modulator frequency. Thus, one laser pulse can be generated for each minimum and maximum value of the change in polarization (i.e., phase difference) imparted by the modulator. Optionally, the pulse is generated at a particular time in the modulator period to coincide with a particular change in polarization (i.e., phase difference). Optionally, the particular change in polarization (i.e., phase difference) is a maximum and minimum change in polarization (i.e., phase difference). Optionally, these particular changes in polarization (i.e., phase difference) correspond to particular resulting polarization states, for example, maximum left-handed and right-handed circular and / or linear polarization states.

[0021] Optionally, the laser repetition rate is at least four times the modulator frequency. Optionally, the laser has a high duty cycle (e.g., greater than 10%) to provide a high signal, especially when used in combination with a lock-in amplifier. The value of the applied phase difference defines the change in polarization of the laser beam.

[0022] Optionally, the reference signal and the difference signal (also called balance signal) are acquired at the laser repetition rate and / or the modulator frequency. The information used for the VCD measurement is especially contained in the modulator frequency component. In particular, the difference signal contains all the information present in the sample signal, except for the noise. In particular, the reference signal is acquired to be used as an offset of the difference signal acquired at the laser frequency. This ensures that the intensity signal of the single channel can be accurately reconstructed. Alternatively, the reference signal and the difference signal are acquired (in particular digitized) with an appropriate resolution and speed, in particular at least one sampling point acquired for each laser pulse, preferably at least three or at least five sampling points acquired for each laser pulse. Optionally, the signal can be averaged for a part of the duration of the pulse or for the entire duration of the pulse. In particular, the VCD spectrum is determined from the recorded pulse, taking into account the modulator amplitude, the modulator phase, and / or the phase difference (delay) induced by the modulator. Optionally, this can be implemented using a lock-in detector, or digital signal processing, or a field programmable gate array (FPGA).

[0023] Optionally, each period of the periodically oscillating change in polarization (i.e., optical delay) imparted by the modulator includes a maximum and a minimum of the imparted change in polarization (i.e., phase difference), and a pulse of the laser beam is generated at a specific time during the periodic oscillation of the modulator (i.e., at a specific time during the modulation period). Optionally, the specific time is selected to coincide with a maximum and a minimum of the imparted change in polarization. Optionally, the specific time is selected to coincide with a maximum imparted circular polarization and / or a maximum imparted linear polarization. Optionally, one pulse of the laser beam is generated for each maximum of the imparted phase difference and one pulse of the laser beam is generated for each minimum of the imparted phase difference. Optionally, the laser repetition frequency is synchronized to the modulator frequency such that the laser operates at the second harmonic of the modulator frequency. The delay between the laser pulse and the modulator period is then adjusted such that the laser pulse is transmitted through the modulator when its change in polarization (i.e., phase difference) corresponds to a maximum circular polarization. This allows the laser power to be fully used for each "perfect" circular polarization state (where "perfect" refers to a completely right-handed and a completely left-handed circular polarization state). Moreover, this scheme is also useful when 100% circular polarization is not reached. Also, this scheme is useful when adjustment of the modulator amplitude should be avoided, but linear polarization should still be minimized. In this case, the maximum phase difference (i.e., the modulator amplitude) is set (high enough) to reach circular polarization at all wavelengths to be measured. This means that for some wavelengths, the maximum phase difference already goes beyond circular polarization and back to linear polarization in the other direction. In this case, the pulses may not coincide with the maximum and minimum of the change imposed on the polarization, but rather with a fraction of the maximum and minimum (e.g., more than 50% but less than 100%). Optionally, the reference signal is acquired at twice the modulator frequency (i.e., in one variant, the laser repetition frequency) and the difference signal is acquired at the modulator frequency and at twice the modulator frequency. This is advantageous.This is because the difference signal specifically conveys the information present in the sample channel (i.e. the VCD signal in this case), while the reference signal only conveys information regarding the intensity of the laser and therefore only needs to be acquired at the laser frequency.

[0024] Optionally, the method further comprises j) transmitting the reference signal and the difference signal to a data acquisition unit (18) and referencing the reference signal and the difference signal to the laser repetition frequency and / or the modulator frequency. Referencing the reference signal and the difference signal to the laser repetition frequency and / or the modulator frequency refers to demodulating the reference signal and the difference signal at the laser repetition frequency and / or the modulator frequency. Optionally, the intensity of the sample light and the intensity of the reference light are repeatedly measured and synchronized with the laser repetition frequency and / or the modulator frequency.

[0025] Optionally, the method includes reducing the beam diameter of the sample light using a telescope system before interacting the sample light with the sample, preferably before modulating the sample light. The telescope system optionally includes a Galilean telescope. Under a Galilean telescope, it is understood that the optical system comprises a converging lens (particularly a plano-convex or concave-convex lens) and a diverging lens (particularly a plano-concave lens). Optionally, the beam diameter of the sample light (at the sample detector) is reduced to less than 5 mm, optionally even less than 2 mm, and optionally even less than 1 mm. The reduced beam diameter light is in particular directed to a tilted photoelastic modulator, the reduction of the beam diameter ensuring separation of the transmitted and reflected beams. The reflected beams result from partial reflections on the optical surfaces of the modulator, and by separating the reflected beams, interference fringes can be avoided.

[0026] A telescope system can reduce the presence of interference fringes on the modulator. Interference fringes occur whenever a beam is reflected off a surface and interferes with itself. By making the beam thinner and positioning the optical elements at an angle, the reflection will be slightly offset from the incident beam. If the beam diameter is made small enough, especially by positioning an aperture or razor blade, the reflected beam will be blocked but the original beam will pass through unblocked. In this way interference cannot occur.

[0027] Optionally, the method comprises the step of k) attenuating the sample light and / or the reference light so that their intensities are closer to each other and / or so that their intensities are within the dynamic range of the sample detector measuring the intensity of the sample light and the reference detector measuring the intensity of the reference light. Optionally, the sample light and / or the reference light are attenuated before interaction with the sample / reference and / or before the modulator and / or before the telescope system. Optionally, the sample light and / or the reference light are attenuated so that their intensities on the respective detectors are closer to each other. Preferably, the sample light and / or the reference light are attenuated so that, when they reach the corresponding detector, their respective intensities on the corresponding detector differ by less than 5%, optionally less than 3% of the total laser power. Small intensity differences of the lasers are preferred. Preferably, the sample light and / or the reference light are attenuated by reflective optical elements in the respective optical paths. The reflective optical element may in particular be a wedge-shaped (e.g. 0.5°) window (e.g. BaF2, CaF2, ZnSe, ZnS) sputtered with a metal (e.g. gold). Optionally, the attenuation is not constant across the spectrum. Preferably, the attenuation is greater in parts of the spectrum where the sample shows weak absorption and / or where the laser has a higher intensity.

[0028] Optionally, the above steps are repeated for further wavelengths to record the (VCD) spectrum of the analyte. In particular, steps a) to h) and optionally steps i), j) and / or k) are repeated using a laser beam generated at a further wavelength to record the spectrum of the analyte. For example, the laser can be tuned to a specific wavelength or in a sweeping mode where the laser wavelength is changed continuously over a series of wavelengths (e.g., 1780 cm). -1 ~1550cm -1 A spectrum can be acquired over a wavelength range of 1000 Hz, e.g., a scan speed of 640 cm / sec). Optionally, the drive voltage of the modulator (especially the EOM) is continuously adjusted over the course of the spectrum acquisition (i.e., the drive voltage of the modulator is adjusted according to the current wavelength). This allows the modulator to be kept in a constant (perfect) quarter-wave state over the entire spectral range. This step only slightly extends the measurement time, since the modulator (especially the EOM) requires a short settling time after each voltage switch. At the same time, the effect of polarization-dependent artifacts can be reduced by continuously adjusting the phase delay over the wavelength range. Optionally, the voltage switches can be performed according to an established relationship between voltage, wavelength, and phase difference. This relationship may be determined from theoretical considerations or from experimental characterization of the modulator.

[0029] Optionally, the modulation of the polarization of the sample light is adjusted to achieve a specific modulation of polarization, preferably for each wavelength in the spectrum.

[0030] Returning to the apparatus, the apparatus preferably comprises a recovery unit configured to recover the intensity variation that depends on the polarization of the sample light from the difference signal.

[0031] Optionally, the apparatus comprises a sample light attenuator for attenuating the sample light and a reference light attenuator for attenuating the reference light, in particular so that each of the sample light and the reference light falls within a dynamic range of a respective detector, the sample light attenuator and the reference light attenuator being optionally reflective attenuators. Optionally, an additional attenuator can be inserted between the laser and the beam splitter.

[0032] Optionally, the apparatus comprises a telescope system in the optical path of the sample light before the sample cell for reducing a beam diameter of the sample light, the telescope system optionally comprising a Galilean telescope.

[0033] Optionally, the sample cell and the reference cell are provided by double pass transmission cells.

[0034] Optionally, an optical path length of the sample light in the sample cell is equal to an optical path length of the reference light in the reference cell. Optionally, an optical path length of the sample light in the sample cell differs by less than 2% compared to an optical path length of the reference light in the reference cell.

[0035] Optionally, a sample light focusing lens is provided for focusing the sample light passing through the modulator and through the sample cell onto the sample detector, and a reference light focusing lens is provided for focusing the reference light passing through the reference cell onto the reference detector.

[0036] Optionally, the modulator comprises a photoelastic modulator (PEM). Optionally, the PEM may comprise ZnSe.

[0037] Optionally, the modulator is disposed in the optical path of the sample light and tilted from a plane perpendicular to the optical path of the sample light, preferably tilted by 5° to 20°, in particular tilted by 15°, relative to said perpendicular plane.

[0038] Optionally, the modulator is tilted around its optical axis oriented at 45° to the vertical. (This disclosure assumes that the linear polarization of the sample light is vertical in particular. This is an arbitrary choice, and changing the polarization to a different direction as required by the optical device is straightforward.) This allows for separation of the reflected beam (i.e., the reference light), thus reducing interference effects at the modulation frequency. The reflected beam can then be blocked by an obstruction or aperture.

[0039] Optionally, the modulator includes an electro-optical modulator (EOM). Preferably, the EOM is positioned orthogonal to the laser beam. Optionally, the EOM operates at a frequency at least an order of magnitude higher than that of commonly used PEM modulators. This allows for increased scanning speed (and therefore lower noise due to the 1 / frequency advantage) while still avoiding interactions between sampling rate and modulator frequency. This allows for improved spectrum per time resolution. The settling time after a voltage (i.e. modulation depth / amplitude) change is significantly shorter for EOMs than for PEMs. This allows for adapting the modulation depth across wavelengths without increasing measurement time.

[0040] Optionally, the optical axis of the modulator (ie, the stress axis in the case of a photoelastic modulator) is oriented at 45° to the intrinsic linear polarization of the laser light source.

[0041] Optionally, a control unit is provided comprising a data acquisition unit connected to the subtractor, connected to the modulator and connected to a waveform generator, the waveform generator being connected to a laser driver of the laser source. Alternatively, instead of being connected to the waveform generator, the data acquisition unit may be (directly) connected to the laser driver. Optionally, the control unit is configured to implement a method according to any of the variants described in the present disclosure. The data acquisition unit may comprise a lock-in amplifier and / or a high-speed analog-to-digital converter and / or an analog sample-and-hold unit. The data acquisition unit may reference the modulator frequency and / or the laser repetition frequency. This can be done directly via the modulator and / or the laser driver, respectively, or indirectly using a waveform generator. In one embodiment (the "free running" embodiment), the laser is not referenced to the modulator. In another embodiment (the "referenced" laser configuration), the laser is referenced to the modulator, for example by using a waveform generator referenced to the modulator. The waveform generator then controls the laser pulse according to a pulse scheme, for example, so that the laser pulse passes through the modulator at the point of maximum amplitude (i.e. maximum and minimum phase difference). "Reference" in this disclosure refers in particular to the synchronization of the waveform generator and the modulator, keeping the phase and frequency between them constant. This can be achieved, for example, by a transistor-transistor logic (TTL) level signal fed into the phase-locked loop of the waveform generator. Optionally, this can also be achieved by controlling both the laser and the modulator with the waveform generator. The delay of the waveform generator may be set so that the pulse corresponds to the maximum / minimum of the sinusoidal modulation period, or to some other defined delay. The detector module may comprise a sample detector, a reference detector, and a subtractor. A difference signal and / or a reference signal may be picked up from the detector module with reference to the laser repetition frequency and the modulator frequency. Optionally, the control unit comprises a restoration unit.

[0042] Optionally, the control unit is configured such that the data acquisition unit acquires data from the subtractor and / or the reference detector and / or the sample detector in synchronization with the laser repetition frequency of the laser light source and / or the modulator frequency of the modulator (in particular in synchronization with both the laser repetition frequency and the modulator frequency).

[0043] Optionally, the modulator is connected to a waveform generator, and the control unit is further configured such that the waveform generator controls the laser source to emit laser pulses depending on the state of the modulator. In this way, the laser source can be controlled to emit laser pulses during a particular modulation state, e.g., passing through the modulator with maximum amplitude.

[0044] The VCD spectrum is defined as the difference in absorption between left-handed circularly polarized light (LHC) and right-handed circularly polarized light (RHC) (see Nafie, LA Vibrational Optical Activity; John Wiley & Sons, Ltd: Chichester, UK, 2011). In particular, the VCD spectrum is determined in the following steps: The sample cell and the reference cell are filled with a solvent. - A reference signal is acquired at the laser frequency (called "reference single channel"). A difference signal is acquired at the laser frequency and at the modulator frequency. - The sample cell is filled with the sample (i.e., solvent and analyte) A difference signal is acquired at the laser frequency and at the modulator frequency.

[0045] The calculation is as follows: The difference signal acquired at the modulator frequency is divided by the sum of the difference signal acquired at the laser repetition frequency and the difference signal acquired in a reference single channel. - This is done for both the solvent and the sample. - The solvent result is then subtracted from the sample result to obtain the VCD spectrum of the sample. Optionally, some corrections can be performed to obtain a more accurate VCD spectrum: "Enantiomer Correction": The two enantiomers are measured (successively). The baseline correction is calculated as the average of the enantiomeric VCD spectra instead of using a solvent. "Racemic correction": Baseline correction using the VCD spectrum of a racemic mixture of sample molecules [Brief description of the drawings]

[0046] The present disclosure will now be further described with reference to figures illustrating advantageous embodiments and variants which should not be construed as limiting the present invention in any way.

[0047] [Figure 1] FIG. 1 is a schematic diagram illustrating a preferred embodiment of an apparatus for circular dichroism analysis of an analyte. [Figure 2A] FIG. 2A is a schematic diagram illustrating an example of a control unit of the apparatus shown in FIG. [Figure 2B] FIG. 2B is a schematic diagram illustrating another example of a control unit of the apparatus shown in FIG. [Diagram 3] FIG. 3 shows the VCD spectrum acquired by the apparatus of FIG. 1 and a comparison of the noise levels of the prior art and the present invention. [Figure 4] FIG. 4 shows the results of long-term measurements of the prior art and the present invention. [Diagram 5] FIG. 5 shows the adjustment of the drive voltage of the modulator as a function of wavelength. [Figure 6A] FIG. 6A shows the sample signal, the reference signal, and the difference signal demodulated at the laser repetition rate. [Figure 6B] FIG. 6B shows an expanded view of the sample signal from FIG. 6A. [Figure 6C] FIG. 6C shows the demodulated sample signal and the difference signal at the modulator frequency. [Figure 7A] FIG. 7(a) shows the spectrum acquired at the laser frequency from the difference signal of the solvent and analyte. [Figure 7B]FIG. 7(B) shows the solvent and analyte spectra after offset correction. [Figure 7C] FIG. 7C shows a conventional absorbance spectrum of the analyte. [Figure 8A] FIG. 8A shows the spectra obtained from the difference signal demodulated at the modulator frequency for the solvent and analyte. [Figure 8B] FIG. 8(B) shows the raw VCD spectrum of the analyte and solvent. [Figure 8C] FIG. 8C shows the solvent-corrected VCD spectrum of the analyte. [Figure 9] FIG. 9 shows a representation of the intensity taken at the laser frequency and the difference in absorbance for left and right circularly polarized light taken at the modulator frequency. [Figure 10A] FIG. 10A shows a representation of the "free running" configuration. [Figure 10B] FIG. 10B shows a representation of the "reference" laser configuration. [Figure 11] FIG. 11 shows a flow chart illustrating an exemplary workflow for VCD measurement using an exemplary apparatus such as that shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] FIG. 1 is a schematic diagram showing a preferred embodiment of a device 1 for circular dichroism analysis of analytes. The device 1 comprises a laser source 2 for providing a laser beam 3. Preferably, the laser source 2 is a tuneable semiconductor laser. The laser beam 3 is directed via a mirror 4 to a beam splitter 5, in particular a 50 / 50 beam splitter. The beam splitter 5 splits the laser beam 3 into a sample light 6a (here the light transmitted through the beam splitter 5) and a reference light 6b (here the light reflected by the beam splitter 5). The sample light 6a is reflected by another mirror 7. A sample light attenuator 8a is provided in the optical path of the sample light 6a for attenuating the sample light 6a, and a reference light attenuator 8b is provided in the optical path of the reference light 6b. The attenuators 8a, 8b are used to adjust the intensity of both beams 6a, 6b so that the intensities of both beams 6a, 6b are similar and fall within the dynamic range of their detectors.

[0049] A telescope system 10 is provided in the optical path of the sample light 6a to reduce the beam diameter of the sample light 6a, in particular to reduce the beam diameter of the sample light to less than 1 mm. The telescope system 10 comprises a Galilean telescope having a converging lens 10a and a diverging lens 10b. A modulator 11 is provided to modulate the polarization of the sample light 6a. The modulator 11 is configured such that its optical axis forms an angle of 45° with the inherent linear polarization of the laser light source 2.

[0050] The inset A of FIG. 1 shows two preferred embodiments of the modulator 11. According to embodiment I, the modulator 11 comprises a PEM 12. The PEM 12 is arranged in the optical path of the sample light 6a, tilted by 15° with respect to a plane orthogonal to the sample light 6a. This allows the separation of the reflected beam and reduces the influence of interference effects. The reflected beam can then be blocked by an occlusion or an aperture. According to embodiment II depicted on the right, the modulator 11 comprises an EOM 13. The EOM 13 is arranged orthogonal to the sample light 6a.

[0051] After the modulator 11, a sample cell 14a is provided for interaction between the sample light 6a and a sample containing an analyte. In addition, a reference cell 14b is provided for interaction between the reference light 6b and a reference. The sample cell 14a and the reference cell 14b are provided by a double path transmission cell 15. The optical path length of the sample light 6a in the sample cell 14a is equal to the optical path length of the reference light 6b in the reference cell 14b.

[0052] The sample detector 15a detects the sample light 6a after interaction with the sample in the sample cell 14a and obtains a sample light signal corresponding to the intensity of the sample light 6a. The reference detector 15b detects the reference light 6b after interaction with the reference in the reference cell 14b and obtains a reference light signal corresponding to the intensity of the reference light 6b. The subtractor 16 forms a difference signal between the sample signal and the reference signal. The balance module 17 includes the sample detector 15a, the reference detector 15b, and the subtractor 16.

[0053] The sample light focusing lens 9a is provided in the optical path of the sample light 6a between the telescope system 10 and the modulator 11, and focuses the sample light 6a via the modulator 11 and the sample cell 14a onto the sample detector 15. The reference light focusing lens 9b is provided in the optical path of the reference light 6b between the reference light attenuator 8b and the reference cell 14b, and focuses the reference light 6b onto the reference detector 15b via the reference cell 14b.

[0054] The device 1 comprises a control unit. Two exemplary embodiments of the control unit (or the electrical configuration and connections of the device 1) are shown in Figures 2A and 2B, respectively. Each of these can be used in conjunction with the embodiment of Figure 1.

[0055] In both embodiments, the controller comprises a data acquisition unit 18, which is connected to the balance module 17, the modulator 11 and a waveform generator 19, which is connected to a laser driver 20 of the laser source 2. Alternatively, the data acquisition unit 18 can be directly connected to the laser driver 20. The laser driver 20 controls the pulsing scheme of the laser source 2. The data acquisition unit 18 can be a lock-in amplifier, a high-speed analog-to-digital converter, or an analog sample-and-hold unit.

[0056] In the embodiment shown in FIG. 2A (also called “free-running” laser configuration), the data acquisition unit 18 references the modulator frequency and the laser repetition frequency. That is, the data acquisition unit 18 acquires data from the balance module 17 (in particular the subtractor 16 and the reference detector 15b) synchronized to the laser repetition frequency of the laser source 2 and synchronized to the modulator frequency of the modulator 11. That is, the data acquisition unit 18 may acquire data from the balance module 17 for every laser pulse of the laser source 2 and every modulation period of the modulator 11. In this embodiment, the laser is not referenced to the modulator. Preferably, the laser repetition frequency is higher than the modulator frequency.

[0057] In the embodiment shown in FIG. 2B (also called the "referenced" laser configuration), a waveform generator 19 is connected to the modulator 11, and the waveform generator 19 (and therefore the pulse scheme of the laser source 2) is referenced to the modulator 11. That is, the waveform generator 19 is controlled by or receives a control signal from the modulator 11 so that the waveform generator 19 can control the pulse scheme according to the modulator frequency. The waveform generator 19 then controls the laser pulse according to the pulse scheme shown in the inset I of FIG. 2B. The delay of the waveform generator 19 is set so that the laser pulse (shown as line 21) corresponds to the maximum and minimum values ​​of the sinusoidal modulation period (shown as line 22). That is, the laser repetition frequency is twice the modulation frequency. As in the embodiment of FIG. 2A, the data acquisition unit 18 acquires the difference signal and the reference signal from the balance module 17 with reference to the laser repetition frequency and the modulator frequency.

[0058] For VCD spectrum measurement, a reference spectrum is first acquired with both cells 14a, 14b filled with solvent (i.e., solvent is used as sample and reference). Then, a background spectrum is acquired again with both cells 14a, 14b filled with solvent (i.e., solvent is used as sample and reference). Then, the sample cell 14a is filled with analyte and a spectrum of the analyte is acquired. The reference spectrum at the laser repetition frequency is added to the balanced channel spectra of both the solvent and the analyte. Then, the VCD spectra of the solvent and the analyte are calculated by dividing the spectrum at the modulator frequency by the spectrum at the laser repetition frequency. The obtained VCD spectrum of the solvent is then subtracted from the VCD spectrum of the analyte to obtain a baseline-corrected VCD spectrum of the analyte.

[0059] Figure 3 shows a typical VCD spectrum acquired with the device shown in Figure 1, where the PEM 12 used as the modulator 11 is operated at 42 kHz, in the "free-running" laser configuration described in the context of Figure 2A. The lower spectrum, labeled "B" for balance operation, corresponds to 0.1 mol of S-1,1'-Bi-2-naphthol (S-BINOL) (i.e., analyte) dissolved in CHCl3 (i.e., solvent) and was acquired over a period of 4:50 min (700 average scans) with the sample cell 14a and the reference cell 14b each having an optical path length of 160 μm. That is, the sample contained analyte and solvent, while the reference contained only solvent and no analyte. The corresponding noise levels are offset and depicted above. "S" shows the noise level of CHCl3 in the 160 μm optical path length cell with 700 average scans when only the sample signal is acquired. This is comparable to classical quantum cascade laser measurement devices. "B" shows the noise level of CHCl3 in a 160 μm path length cell with 700 average scans acquired according to the present embodiment. The noise is reduced by 3.5 times, resulting in smaller variations between different replicates. Compared to the literature, the root mean square error (RMS) is improved by a factor of 4 (in 1 / 6 of the measurement time). The height of the noise level is the maximum deviation from the zero line, which is improved by a factor of 13 (in 1 / 6 of the measurement time).

[0060] In the literature (United States Pharmacopeia and National Formulary (USP 41-NF 36)), the signal to noise level is 5×10 -5 (60 min), whereas in the present invention, the signal-to-noise level was 3.8×10 -6 (10 min). In addition, in the literature (M. Bonmarinand J. Helbing, Opt. Lett., 2008, 33, 2086), the RMS error of the baseline was 8×10 -6 (60 min), whereas in the present invention, the baseline RMS error was 1.9 × 10 -6 (10 minutes).

[0061] The results of long-term measurements are shown in Figure 4. For both the "conventional" setup with a single detector ("S") and the "balanced" setup with two matched MCT detectors ("B"), five acquisitions of the 100% CHCl3 line in a 160 μm cell were performed with a scan average of up to 1500 scans (10 min). For each spectrum, the peak at 1730 cm -1 ~1600cm -1 The root mean square error (RMS) and maximum deviation from zero in the spectral range of 1000 nm were calculated. Deviation is a figure of merit that characterizes VCD spectra according to The United States pharmacopeia. The National formulary. The plotted curves compare the performance of S and B as the average of five measurements, and the error bars represent the standard deviation. The top panel shows the RMS noise versus the average scan / measurement time, and the bottom panel plots the deviation from zero versus the average scan / measurement time.

[0062] FIG. 5 shows the adjustment of the drive voltage of the modulator 11 according to the wavelength. Circular polarization is achieved by a retardation of λ / 4. However, for one defined modulator drive voltage, the exact retardation of λ / 4 is achieved only for one wavelength. This leads to elliptical polarization for the remaining wavelengths when the spectrum is recorded. The QCL emits monochromatic light, but its wavelength is time-dependent. The retardation that the modulator 11 imparts to the sample light 6a is proportional to the drive voltage of the modulator 11. The drive voltage of the modulator 11 is preferably adjusted during the course of the spectrum acquisition (i.e., the drive voltage of the modulator 11 is adjusted according to the current wavelength). This allows the retardation of the sample light 6a of the modulator 11 to be maintained at a constant (perfect) λ / 4 state over the entire spectral range. FIG. 5 shows the retardation and drive voltage imparted with and without this correction.

[0063] Exemplary spectra and approaches to obtaining them are described below.

[0064] Working principle: The balanced detection module (including sample detector 15a, reference detector 15b, and subtractor 16) outputs both the voltage from reference detector 15b and the voltage from sample detector 15a (or the inverted voltage from sample detector 15a, respectively). In addition, the balanced detection module also outputs a difference signal between the two, called the balance channel. This voltage carries information about the conventional absorbance of the analyte at the laser repetition frequency, and the difference in the absorbance of circularly polarized light at the modulator frequency. The resulting spectra are shown in Figures 6A-6C.

[0065] In particular, FIG. 6A shows the reference signal, the sample signal (inverted due to the specific electronic configuration), and the balance channel (i.e., the difference signal). FIG. 6B shows the balance channel enlarged from FIG. 6A. Note the voltage units in FIG. 6B compared to FIG. 6A. FIG. 6A and FIG. 6B are demodulated at the laser repetition rate. FIG. 6C shows the VCD spectrum acquired at the modulator frequency of the sample signal and the balance channel.

[0066] Lock-in amplifier as data acquisition unit: To calculate the conventional absorbance, the intensity of the background and the intensity of the analyte are taken at the laser frequency (Equation 1):

[0067]

number

[0068] The voltage acquired at the laser frequency of the balance channel contains only the intensity difference and must be adjusted with an offset: the voltage acquired at the laser frequency of the reference signal (FIG. 6A), which is acquired once with only solvent in both the sample cell 14a and the reference cell 14b, and added to each balance output (Equation 2).

[0069]

number

[0070] This equation is illustrated in Figures 7A-7C, where Figure 7A shows the spectrum acquired at the laser frequency, Figure 7B shows the offset corrected solvent and analyte spectra, and Figure 7C shows the calculated absorbance of the analyte.

[0071] In particular, Figure 7A shows spectra acquired from the solvent and analyte balance channels at the laser frequency. A reference channel acquired at the laser frequency is also shown, which is used as an offset for the balance channel spectra. Figure 7B shows the solvent and analyte spectra after offset correction. Figure 7C shows the -log 10 1 shows the conventional absorbance spectrum of the analyte, calculated according to (analyte / solvent).

[0072] For a VCD spectrum, the general formula is (Equation 3):

[0073]

number

[0074] Generally, the solvent is chosen to be non-chiral, but the VCD spectrum of the solvent may still differ from zero. This is due to birefringence occurring in the optical device, which may be present in the lenses, the detector windows, the cell, and any optical elements after the modulator. Subtraction of the VCD spectrum of the solvent eliminates the effect of these artifacts. Absorbance artifacts arising from the sample itself may distort the VCD spectrum, in which case baseline correction can be performed by subtracting the spectrum measured for the racemate or the other enantiomer.

[0075] In this exemplary device, JPEG2025507185000011.jpg1315 corresponds to the intensity taken from the balanced channel and demodulated at the modulator frequency. Since JPEG2025507185000012.jpg1118 corresponds to conventional IR intensity, in this device it can be defined as the balanced channel demodulated at the laser frequency and offset corrected (Equation 2, see Figures 7A-7C). Thus, Equation 3 can be rewritten for this device as follows (Equation 4):

[0076]

number

[0077] The two parts are referred to as the raw VCD spectra of the analyte and the solvent, respectively.

[0078] This is illustrated in Figures 8A-8C. In Figure 8A, the spectra sampled from the balanced channel demodulated at the modulator (specifically, PEM) frequency are shown. Using these, the raw VCD spectrum in Figure 8B can be calculated according to Equation 4. Although the solvent (in this example, CHCl3) is achiral, the VCD spectrum deviates from zero and contains artifacts present in the optical train. Therefore, the VCD spectrum of the analyte must be corrected to account for these artifacts by subtracting the VCD spectrum of the solvent. The result is shown in Figure 8C.

[0079] In particular, Figure 8A shows the spectra acquired from the balanced channel demodulated at the modulator frequency for the solvent and analyte, and Figure 8B shows the raw VCD spectra for the analyte and solvent. JPEG2025507185000014.jpg2122. Figure 8C shows the solvent-corrected VCD spectrum of the analyte, calculated by subtracting the spectrum in Figure 8B.

[0080] Figure 9 shows a representation of I and ΔI, where I corresponds to the intensity acquired at the laser frequency, and ΔI corresponds to the difference in absorbance of left and right circularly polarized light acquired at the modulator frequency.

[0081] The above formula is applicable when the laser is used in a "free-running" or "reference" configuration. Both configurations are shown in Figures 10A and 10B. The difference in data acquisition between the "reference" and "free-running" laser configurations is all in the laser frequency. This laser frequency is significantly higher than the modulator frequency (set to 1 MHz, for example) in the "free-running" configuration, but is the second harmonic of the modulator frequency in the "reference" laser configuration.

[0082] In particular, FIG. 10A shows a representation of a "free running" configuration. The laser is not referenced to the modulator, but is set to a higher pulse frequency than the modulator, providing laser pulses for most of the minima / maxima of the modulation period. FIG. 10B shows a "reference" laser configuration, where the laser operates at the second harmonic of the modulator. One pulse is generated for each minimum and maximum of each modulation period. Preferably (but not shown in FIG. 9B), they are offset from the actual maximum and minimum of the modulation period to ensure maximum circular polarization.

[0083] High-speed DAC or sample-and-hold circuit: The "reference" laser configuration provides another option for performing data acquisition. Since the pulses are aligned with "perfect" phase difference, in this case corresponding to circular polarization, they can be digitized separately and used to calculate the absorbance for left and right circular polarization, respectively. Thus, Equation 1 becomes:

[0084]

number

[0085] The VCD spectrum obtained in this case is already baseline corrected with the solvent.

[0086] Modulator Settings: Whatever the configuration, the phase difference imparted by the modulator can be adjusted in parallel with the wavelength sweep, ensuring precise polarization at each wavelength.

[0087] The flow chart in Figure 11 shows the general workflow for one VCD measurement using the balance detection device. Note that the "reference" part only needs to be performed when changing the solvent or at the beginning of a series of measurements.

[0088] The invention is the result of research carried out within the course of the COMET Centre CHASE, which is supported within the COMET (Competence Centers for Excellent Technologies) programme by BMK Austria, BMDW Austria, the States of Upper Austria and Vienna. The COMET programme is managed by the Austrian Research Agency (FFG).

Claims

1. A method for analyzing the vibrational circular dichroism of an analyte, a) A step of generating a laser beam (3) at a wavelength in the infrared region, b) The step of splitting the laser beam (3) into a sample light (6a) and a reference light (6b), c) Modulating the polarization of the sample light (6a), d) A step of interacting the sample containing the analyte with the sample light (6a), e) The step of making the reference light (6b) and the reference interact, f) A step of measuring the intensity of the sample light (6a) and obtaining a sample signal, g) A step of measuring the intensity of the reference light (6b) and obtaining a reference signal, h) A step of obtaining a difference signal by subtracting the sample signal from the reference signal, Methods that include...

2. i) The method according to claim 1, further comprising the step of reconstructing the polarization-dependent intensity change introduced by the analyte from the difference signal.

3. The method according to claim 2, wherein the change in the intensity of the polarization dependence is due to the difference in absorption of left-handed and right-handed circularly polarized light in the sample or analyte.

4. The method according to claim 1 or 2, wherein each of the intensity signal based on the reference signal and the intensity signal based on the sample signal is calculated from the difference signal and the reference signal and / or the sample signal.

5. The method according to claim 4, wherein the polarization-dependent intensity change introduced by the analyte from the difference signal is scaled using an intensity signal based on the reference signal or an intensity signal based on the sample signal.

6. The laser beam (3) is generated by pulses having a laser repetition frequency, The sample light (6a) is modulated by a modulator (11) that changes the polarization according to the modulator frequency. The laser repetition frequency is equal to or greater than the modulator frequency. The method according to claim 1.

7. Each period of the periodically oscillating change in the polarization applied by the modulator (11) includes the maximum and minimum values ​​of the change applied to the polarization. The pulse of the laser beam (3) is generated at a specific time during the periodic oscillation of the modulator (11). The method according to claim 6.

8. The method according to claim 7, wherein the specific time is selected to coincide with the maximum and minimum values ​​of the change imparted to the polarization.

9. The method according to claim 7, wherein the specific time is selected to coincide with the maximum circular polarization and / or the maximum linear polarization to be imparted.

10. j) The method according to claim 6, further comprising the step of transmitting the reference signal and the difference signal to a data acquisition unit (18) and causing the reference signal and the difference signal to reference the laser repetition frequency and / or the modulator frequency.

11. Before the sample light (6a) interacts with the sample, the telescope system (10 The method according to claim 1, comprising reducing the beam diameter of the sample light (6a) using ).

12. k) The method according to claim 1, further comprising the steps of attenuating the sample light (6a) and / or the reference light (6b) such that their intensities are closer to each other, and / or attenuating their intensities so that they fall within the dynamic range of a sample detector (15a) for measuring the intensity of the sample light (6a) and a reference detector (15b) for measuring the intensity of the reference light (6b).

13. The method according to claim 1, wherein steps a) to h) are repeated using the laser beam (3) generated at a further wavelength in order to record the spectrum of the analyte.

14. The method according to claim 13, wherein the polarization modulation of the sample light (6a) is adjusted to achieve a specific polarization modulation for each wavelength in the spectrum.

15. Apparatus (1) for vibrational circular dichroism analysis of an analyte, - A laser light source (2) for supplying the laser beam (3), - A beam splitter (5) for splitting the laser beam (3) into a sample light (6a) and a reference light (6b), - A modulator (11) for modulating the polarization of the sample light (6a), - A sample cell (14a) for the interaction between the sample light (6a) and the sample containing the analyte, - A reference cell (14b) for the interaction between the reference light (6b) and the reference, - A sample detector (15a) for detecting the sample light (6a) and obtaining a sample signal corresponding to the intensity of the sample light (6a), - A reference detector (15b) for detecting the reference light (6b) and obtaining a reference signal corresponding to the intensity of the reference light (6b), - A subtractor (16) for forming a difference signal between the sample signal and the reference signal, A device (1) equipped with the following:

16. The apparatus (1) according to claim 15, comprising a sample light attenuator (8a) for attenuating the sample light (6a) and a reference light attenuator (8b) for attenuating the reference light (6b), wherein each of the sample light (6a) and the reference light (6b) is attenuated so that it falls within the dynamic range of the corresponding detector, and the sample light attenuator (8a) and the reference light attenuator (8b) are reflective attenuators.

17. The apparatus (1) according to claim 15 or 16, wherein a telescope system (10) is provided in the optical path of the sample light (6a) in front of the sample cell (14a) in order to reduce the beam diameter of the sample light (6a), and the telescope system (10) comprises a Galilean telescope.

18. The apparatus (1) according to claim 15 or claim 16, wherein the sample cell (14a) and the reference cell (14b) are provided by a double-pass transmission cell.

19. The apparatus (1) according to claim 15 or claim 16, wherein the optical path length of the sample light (6a) in the sample cell (14a) is equal to the optical path length of the reference light (6b) in the reference cell (14b).

20. The sample light (6a) is passed through the modulator (11) and the sample cell (14a). The apparatus (1) according to claim 15 or claim 16, further comprising: a sample light focusing lens (9a) for focusing light onto the sample detector (15a); and a reference light focusing lens (9b) for focusing the reference light (6b) onto the reference detector (15b) through the reference cell (14b).

21. The apparatus (1) according to claim 15 or claim 16, wherein the modulator (11) includes a photoelastic modulator (12).

22. The apparatus (1) according to claim 15 or claim 16, wherein the modulator (11) includes an electro-optic modulator (13).

23. The apparatus (1) according to claim 15 or claim 16, wherein the modulator (11) is positioned in the optical path of the sample light (6a) which is tilted from a plane perpendicular to the optical path of the sample light (6a), and is positioned at an angle of 5° to 20° or 15° with respect to the plane perpendicular to the optical path of the sample light (6a).

24. The apparatus (1) according to claim 15 or claim 16, wherein the optical axis of the modulator (11) is oriented at a 45° angle with respect to the intrinsic linear polarization of the laser light source (2).

25. The apparatus (1) according to claim 15 or claim 16, comprising a control unit including a data acquisition unit (18) connected to the subtractor (16), the modulator (11), and the waveform generator (19), wherein the waveform generator (19) is connected to the laser driver (20) of the laser light source (2).

26. The apparatus (1) according to claim 25, wherein the control unit is configured such that the data acquisition unit (18) acquires data from the subtractor (16) and / or the reference detector (15b) and / or the sample detector (15a) which are synchronized with the laser repetition frequency of the laser light source (2) and / or the modulator frequency of the modulator (11).

27. The apparatus (1) according to claim 26, wherein the modulator (11) is connected to the waveform generator (19), and the control unit is further configured so that the waveform generator (19) controls the laser light source (2) to emit laser pulses according to the state of the modulator (11).

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