Dimension selective optical spectroscopy

Abstract

A spectroscopic method and system for simultaneous analysis of the molecular structure and size of a single compound or a mixture (M) of different molecules (M1, M2). A liquid volume (V) with a mixture (M) is provided, the mixture (M) having an initial concentration difference ([delta] M) between different sub-volumes (Vm, Vs) fluidly interconnected and arranged in different positions along a spatial coordinate (X). A time-dependent spectrum (alpha [t, vj]) of light (L) interacting with a partial liquid volume (V) is measured while the concentration difference ([delta] m) is at least partially balanced by diffusion of different molecules (M1, M2) between different sub-volumes (Vm, Vs) along spatial coordinates (X). The time correlation spectrum (alpha [t, vj]) comprises respective different spectral profiles (alpha 1, alpha 2) of respective different molecules (M1, M2). Each spectral profile ([alpha] 1, [alpha] 2) may have a different time-dependent evolution ([tau] 1, [tau] 2) in a time-dependent spectrum ([alpha] [t, [nu] j]) caused by a respective different diffusion characteristic (D1, D2) of a different molecule (M1, M2).

Description

Technical Field

[0001] The present disclosure relates to spectroscopic methods and systems for simultaneously analyzing the chemical structure and size of a compound or a mixture of compounds having different molecular or particle sizes. Background Art

[0002] Identification and / or characterization of molecular structure and size is important in many fields, ranging from daily practice in synthesis laboratories to biomedicine and industry. Despite the availability of many techniques, simultaneous characterization of structure and size of chemical species remains challenging in many cases, especially in mixtures.

[0003] On the one hand, there are various size selection techniques, which usually have specific application areas. For some applications, gas chromatography can be used to separate and analyze compounds that can evaporate without decomposition. For other or additional applications, dynamic light scattering can be used to determine the size distribution curve of small particles in a suspension or polymer solution. For other or additional applications, gel electrophoresis can be used to separate and analyze macromolecules and their fragments based on the size and charge of macromolecules (such as DNA, RNA and proteins). Unfortunately, the information on the molecular structure itself provided by these and other size selective techniques may be limited. As a partial solution, nuclear magnetic resonance diffusion ordered spectroscopy (NMR-DOSY) can be used to characterize mixed compounds. However, NMR-DOSY requires NMR spectrometer equipment, and in many cases, such as paramagnetic compounds, compounds with insufficient NMR signals, or compounds with complex and / or overlapping NMR spectra, this technology is not applicable.

[0004] On the other hand, there are various spectroscopic techniques that are widely used by measuring how different forms of light interact with atoms and molecules. Typically, these spectroscopic techniques involve the measurement of the (wavelength-dependent) absorption, emission, reflection and / or scattering of a light beam irradiating a sample. In many applications, infrared (IR) spectroscopy (also called vibrational spectroscopy) is used to measure the interaction of a substance with infrared light (e.g. between 0.8-250 μm, up to 1 mm) and / or mid-infrared light (typically between 2.5-25 μm, i.e. 4000-400 cm -1 ) interactions. For example, (linear) IR absorption spectroscopy can be used to measure the corresponding vibrational modes of various molecules. Other or additional related techniques, such as Raman spectroscopy and (vibrational) circular dichroism spectroscopy, can also be used. Other or additional linear or nonlinear techniques, such as two-dimensional pump-probe or photon echo spectroscopy, etc. can also be used. Various spectroscopic techniques can also be used with other or additional wavelengths of light, such as ultraviolet and / or visible spectroscopy (UV-Vis).

[0005] These and other spectroscopic techniques based on light-matter interactions can be advantageously used to study and identify chemical substances or functional groups in solid, liquid or gaseous form. For example, advantages of using known spectroscopic techniques to characterize various molecules may include that these techniques are easy, simple, non-invasive, label-free, and that peaks in the spectrum provide direct information on the chemical structure and conformation of the compound. However, spectroscopic techniques are generally insensitive to molecular / particle size; and in the case of mixtures, known spectroscopic techniques also have unfortunate limitations, such as overlap of spectral signatures with relatively broad peaks, particularly in liquid solutions, which can make it difficult to distinguish peaks and / or identify compounds. These and other limitations have so far limited the use of known spectroscopic techniques to characterizing molecular size and directly analyzing mixtures.

[0006] Therefore, there remains a need for further improved spectroscopic methods and systems for simultaneous analysis of molecular structure and size, particularly in various mixtures, e.g. to mitigate the limitations of known spectroscopic techniques while maintaining at least some of their advantages. Summary of the invention

[0007] Various aspects of the present disclosure relate to methods and systems for spectroscopic analysis of molecular structure and / or size, in particular of the molecular structure and / or size of a mixture of different molecules. A liquid volume with a mixture is provided, such as a liquid volume formed in a container or a sample cell, such that initially there is a concentration difference of the molecules between different interconnected subvolumes of the liquid volume. While the concentration difference is at least partially balanced by diffusion of the different molecules between the different subvolumes, a time-correlated spectrum of light interacting with a corresponding measurement position of the corresponding subvolume is measured. Thus, the time-correlated spectrum may include spectral signatures of different molecules, wherein each spectral signature may have a different time-correlated evolution in the time-correlated spectrum, which is caused by the corresponding different diffusion characteristics of the different molecules.

[0008] As a basic principle, it is recognized that there is a correlation between the diffusion coefficient "D" of a particle (e.g., a molecule or molecular aggregate) and its size "R". This can be modeled using the Stokes-Einstein relationship. According to this relationship, large particles diffuse more slowly than small particles. Therefore, the change in spectral intensity measured at a specific location in a sample with an initial concentration gradient can be related to the size of the corresponding particle and / or molecule that diffuses from one location to another (D ~ 1 / R, the proportionality constant is determined by the solvent viscosity). This principle can be used to simultaneously determine the size and spectrum of species present in a mixture of different molecules.

[0009] In an advantageous application of the present teachings, the inventors have designed a liquid sample cell that makes it possible to separate spectra of different molecules in a mixed solution. These and other devices can be used to generate multidimensional spectra in which different molecules can be separated based at least on their diffusion properties.

[0010] For specific applications in the infrared domain, the inventors refer to the present technology as Diffusion Ordered-InfraRed Spectroscopy ("IR-DOSY"). For example, a two-dimensional IR-DOSY spectrum can simultaneously provide IR spectra and diffusion constants (and therefore sizes) of different compounds in a mixture. Compared to other size-selective techniques, the IR vibration frequencies in the IR-DOSY spectrum provide direct information on the chemical structure. Of course, it will be recognized that although IR-DOSY spectra may look similar to NMR-DOSY spectra at first glance, the working principles of IR and NMR-DOSY are completely different. In addition, compared to existing methods, the advantages of IR-DOSY may include ease, simplicity, non-invasiveness, and the absence of any sample preparation, and does not have some of the disadvantages of NMR, for example, as noted in the background technology section above.

[0011] In a preferred embodiment as described herein, the system includes a specially constructed liquid cell for IR transmission spectroscopy, and a standard syringe pump to inject solutions and solvents. Most preferably, the cell is suitable for the standard liquid cell mount of any standard FTIR (Fourier transform IR) spectrometer. Alternatively or additionally, an IR microscope can also be used to scan the sample, as described herein. Using a syringe pump, a sample solution (M) can be injected into one inlet and a pure solvent (S) can be injected into another inlet. Specifically, the sample solution (M) and its solvent (S) are injected into the narrow space between two infrared transparent windows so that the two liquids are in contact with a constant parallel flow. After the pumping stops (at T = 0), the dissolved compound can diffuse into the solvent-filled half of the cell, and the rate depends on the size of the dissolved compound. Time-dependent infrared absorption spectra can be measured, for example, at a position in the sample space where there is only solvent at the beginning. Over time, solute molecules begin to appear in the IR spectrum, and the spectral intensity increases at different rates depending on the size of the compound. Since the IR beam in a standard FTIR spectrometer typically has a diameter of several millimeters, the IR spectrum of a specific area of ​​the cell can be selected by an adjustable optical slit (e.g., with a sub-millimeter width). Other spatial selections can also be used, such as focusing the beam at a specific location in the sample cell.

[0012] The present teaching thus provides a user-friendly and simple way to simultaneously characterize the size and chemical structure of molecules, molecular aggregates / complexes and small particles (of course also formed by molecules). The adjustable slit position and width make it possible to adjust the size range sampled by the device: particles of larger sizes (e.g. nanoplastics) may take a relatively long time to diffuse into the measurement area, but the measurement time can be shortened arbitrarily, for example by moving the slit closer to the liquid-liquid contact line between the sample solution and the solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] These and other features, aspects, and advantages of the apparatus, systems, and methods of the present disclosure will become better understood from the following description, appended claims, and accompanying drawings, in which:

[0014] Figures 1A to 1C shows the measurement of a mixture of different molecules during equilibrium of concentration differences;

[0015] FIG. 2A to FIG. 2C The measurement and processing of time-correlated spectra are shown;

[0016] FIG. 3A to FIG. 3C shows the initial concentration differences between the different subvolumes provided by a set of parallel flows;

[0017] FIG. 4A to FIG. 4C illustrates aspects of a sample cell configured to provide parallel flow in an elongated flow channel;

[0018] Figure 5A Further aspects of the sample cell are shown;

[0019] Figure 5B A system for spectroscopic analysis of a mixture is shown;

[0020] Fig. 6A and Figure 6B A photograph showing a positioning device configured to adjust a measurement position in a sample cell;

[0021] Fig. 7A Infrared microscope images showing parallel flows in an elongated flow channel;

[0022] Figure 7B and Figure 7C Infrared microscope images at different times after the flow has stopped are shown;

[0023] Fig. 8A shows a time-dependent infrared absorption spectrum for measuring a mixture of different molecules diffused into the field of view of the measurement location;

[0024] Figure 8B The corresponding diffuse order spectrum is shown;

[0025] Fig.9A and Fig. 9B Additional spectrograms are shown;

[0026] Fig.10 A two-dimensional spectrum of the mixture based on subsequent scans is shown;

[0027] FIG. 11A to FIG. 11C It is shown that providing a liquid volume containing a mixture having an initial concentration difference by removing barriers between different subvolumes;

[0028] FIG. 12A to FIG. 12C showing a liquid volume in which the mixture has an initial concentration difference between a first subvolume to which the mixture is added and a second subvolume including a measurement location;

[0029] Fig.13A and Fig. 13B An experiment using relatively large channel thickness is shown. DETAILED DESCRIPTION

[0030] The terms used to describe specific embodiments are not intended to limit the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms "one", "a kind of" and "the" are intended to also include plural forms. The term "and / or" includes any and all combinations of one or more related listed items. It should be understood that the terms "include" and / or "comprise" specify the presence of the features, but do not exclude the presence or addition of one or more other features. It will be further understood that, unless otherwise stated, when a particular step of a method is referred to as being after another step, it may directly follow the other step, or one or more intermediate steps may be performed before performing the particular step. Similarly, it should be understood that when describing a connection between a structure or component, unless otherwise stated, the connection may be established directly or through an intermediate structure or component.

[0031] The present invention is described more fully below with reference to the accompanying drawings, in which embodiments of the invention are shown. In the accompanying drawings, the absolute and relative sizes of systems, components, layers and regions may be exaggerated for clarity. Embodiments may be described with reference to schematic and / or cross-sectional illustrations of embodiments and intermediate structures of the invention that may be idealized. In the specification and drawings, like reference numerals always refer to like elements. Relative terms and their derivatives should be interpreted as referring to the orientation described at the time or the orientation shown in the figure being discussed. These relative terms are for ease of description and do not require the system to be constructed or operated in a specific orientation unless otherwise specified.

[0032] Figures 1A to 1CA mixture "M" of different molecules M1, M2 is shown being measured during equilibrium concentration difference ΔM. In some embodiments, for example, as shown, a liquid volume "V" having a mixture "M" (i.e., the molecules to be analyzed) is provided, the mixture "M" having an initial concentration difference ΔM between different subvolumes Vm, Vs of the liquid volume "V". In one embodiment, the different subvolumes Vm, Vs are fluidically connected, such as directly adjacent, preferably across a liquid interface "I". Thus, the subvolumes Vm, subvolumes Vs may be integrally connected and / or form a continuous liquid volume "V". In another or further embodiment, the different subvolumes Vm, Vs are arranged at different positions along at least one spatial coordinate "X". In other or further embodiments, while the concentration difference ΔM is at least partially balanced by (translational) diffusion of different molecules M1, M2 between the different subvolumes Vm, Vs along the spatial coordinate "X", a time-dependent spectrum of light interacting with the liquid volume "V" is measured at a measurement position "P" in (at least) one of the different subvolumes Vm, Vs.

[0033] FIG. 2A to FIG. 2C The processing of a time-correlated spectrum α(t, v) obtained, for example, according to the methods and systems described herein is shown. For example, the time-correlated spectrum α(t, v) may include one or more of an infrared spectrum, a visible spectrum, a UV spectrum, an absorption spectrum, a transmission spectrum, a reflection spectrum, a scattering spectrum, a Raman spectrum, etc. In some embodiments, the time-correlated spectrum α(t, v) includes corresponding different spectral feature graphs α1, α2 of corresponding different molecules M1, M2. In other or further embodiments, each spectral feature graph α1, α2 has a different time-correlated evolution τ1, τ2 in the time-correlated spectrum α(t, v), which is caused by the corresponding different diffusion characteristics D1, D2 of the different molecules M1, M2.

[0034] In some embodiments, the mixture "M" is analyzed based on the time-correlated spectrum α(t, v). In one embodiment, analyzing the mixture "M" includes identifying one or more of the different molecules M1 and M2 in the mixture "M" based on the time-correlated spectrum α(t, v). For example, different molecules M1 and M2 can be identified based on a combination of spectral features in the corresponding spectral feature graphs α1 and α2. In another or further embodiment, analyzing the mixture "M" includes determining one or more (absolute or relative) corresponding concentrations of different molecules M1 and M2 in the mixture "M". For example, the concentration can be derived using a reference or known spectral intensity of the corresponding spectral feature graphs α1 and α2. In another or further embodiment, analyzing the mixture "M" includes determining the molecular properties of one or more of the different molecules M1 and M2 in the mixture "M", such as molecular structure and / or configuration. For example, using infrared spectroscopy, various vibration modes of different molecules M1 and M2 can be measured. In another or further embodiment, analyzing the mixture "M" includes determining the frequency (or wavelength) and / or amplitude of the spectral features (e.g., absorption peaks) in one or more spectral feature graphs α1, α2 of different molecules M1, M2. Other or additional (such as known or yet to be discovered) spectral analysis techniques may also be used.

[0035] In some embodiments, the time-correlated spectrum α(t,v) is decomposed to determine (e.g., separate) corresponding different spectral signatures α1, α2 of different molecules M1, M2, and / or to determine corresponding different diffusion characteristics D1, D2 of different molecules M1, M2. In other or further embodiments, the spectral analysis of the mixture "M" includes identifying one or more molecules M1, M2 in the mixture "M" based on a priori known spectral signatures α1, α2 of different molecules and / or based on a priori known diffusion characteristics D1, D2 of different molecules. For example, identification includes finding the molecule with the closest matching spectral signature and / or the closest matching diffusion characteristic for each component in the time-correlated spectrum α(t,v). It will be appreciated that even matching only the spectral signatures (which can be better separated using diffuse ordered spectra) can provide significantly improved identification, for example when compared to conventional spectra. In addition, this can be improved by alternatively or additionally matching the measured diffusion characteristics.

[0036] In some embodiments, the mixture "M" includes nano-scale and / or micro-scale particles. For example, each particle can include a single molecule, or the particles can be formed by aggregates of the same or different molecules. Typically, the size or diameter of such particles can range from less than one nanometer to one hundred micrometers, or even to one thousand micrometers or more. For example, the average size or diameter of protein aggregates can be less than one micrometer, less than one hundred nanometers, or even less than ten nanometers. For example, the average size or diameter of micron-scale particles can be greater than one hundred nanometers, greater than one micrometer, greater than ten nanometers, or even greater than one hundred nanometers, such as up to one millimeter or more.

[0037] Possibly, some or all particles in the mixture "M" may have substantially the same or similar spectral features and / or overlapping spectral peaks, but have different sizes. For example, the mixture "M" includes molecules and / or particles, which may include one or more of the following: molecular ions, molecules, molecular complexes, supramolecular structures, polymers, protein aggregates, (self-assembled) protein fibrils, amyloid proteins, transition metal complexes, plastic particles, etc. For these and other embodiments, the diffusion properties can provide, for example, key information about the (relative and / or absolute) size of the particles, which is difficult or impossible to measure otherwise, especially in (natural) liquid solutions. For example, the present teachings can be advantageously applied to measuring water contamination by nanoscale and / or microscale (plastic) particles in, for example, seawater, river water, lake water, rainwater, etc.

[0038] Without being bound by theory, the measured time-dependent spectrum α(t, v) can generally be represented by a matrix of measurement data. For example, each row (or column) of the matrix can be formed by the corresponding spectrum measured at a particular time. Generally, such a matrix can be decomposed into different spectral components and / or time components. In some embodiments, the matrix can be decomposed and written as the inner product of the columns of the corresponding time-dependent evolutions τ1, τ2 (e.g., relative concentration as a function of time "t") and the corresponding rows of the spectral signatures α1, α2 (e.g., spectral intensity as a function of wavelength or frequency "v"):

[0039]

[0040] Of course, rows and columns can also be interchanged and / or swapped. In principle, decomposition is more feasible when the time-dependent evolutions τ1, τ2 are more linearly independent, for example when the diffusion characteristics D1, D2 are significantly different; and when the spectral signatures α1, α2 are more linearly independent, for example when different molecules have significantly different spectral signatures (e.g. different absorption peaks). For example, the linear independence and / or contribution of different components can be determined based on a singular value decomposition (SVD) of the matrix, for example based on the singular values ​​obtained from such decomposition. Of course, other data analysis methods can also be used. As an alternative to measurement data forming a two-dimensional matrix, measurement data stored in a matrix of more than two dimensions can also be used, for example, one or more additional parameters vary along a third dimension or another dimension. For example, a sub-portion of a higher dimensional matrix can be used, or a higher dimensional matrix can be processed in any other way.

[0041] In some embodiments, decomposition is achieved by modeling the time-dependent evolutions τ1, τ2 and fitting the spectral feature maps α1, α2, or vice versa. For example, the time-dependent evolutions τ1, τ2 can be modeled according to a diffusion model using one or more diffusion characteristics D1, D2 as parameters. Other variable or invariant parameters may also be included, such as the distance Xp and / or the range of the distance ΔX between the initial interface "I" and the measurement position "P". In one embodiment, the time correlation is modeled using a formula obtained by solving one or more diffusion equations. In another or further embodiment, a mathematical process is used to transform the time-dependent axis of the two-dimensional data set into a diffusion constant axis (or equivalently, a size axis). For example, the mathematical process may include an inverse Laplace transform or PCA combined with a global fit.

[0042] In one embodiment, the time-dependent concentrations of the respective molecules that form the particles diffusing through the liquid volume are modeled based on the respective diffusion properties (e.g., the respective known or unknown diffusion coefficients "D"). For example, the time-dependent concentrations of each different molecule can be modeled using a diffusion equation, which can be written as:

[0043]

[0044] Wherein the value of D depends on the molecular size. The time- and position-dependent concentrations can be derived based on the distance Xp (with an optional range ΔX) between the origin of the different molecules M1, M2 at T0 and the measurement position "P". For example, the diffusion equation models the different time dependencies of the concentrations of the different molecules M1, M2 at the measurement position "P". Of course, other diffusion modeling can also be used.

[0045] In another or further embodiment, the diffusion coefficient "D" is modeled according to the Stokes-Einstein equation, for example written as:

[0046]

[0047] Among them, "k B ” is Boltzmann's constant; “T L " is the absolute temperature of the liquid; "η" is the dynamic viscosity of the liquid; and "R" is the radius of the equivalent spherical particle. It will be appreciated that the measured diffusion coefficient for an unknown molecule can also be used to estimate the corresponding molecular mass and / or size, for example, assuming that this is related to the radius "R". If necessary, the measured diffusion coefficient for different temperatures "T L ” and / or different solvents (to affect the dynamic viscosity). Of course, other or additional modeling of diffusion coefficients may also be used, such as the Einstein-Smoluchowski equation for the diffusion of charged particles in an externally applied electric field. Thus, some embodiments may include determining at least one of the respective sizes, masses and / or charges of different molecules M1, M2 based on the measured diffusion characteristics D1, D2 and / or based on their respective modeling.

[0048] In some embodiments, for example, Figure 1A and Figure 1B As shown, between a first position in the liquid volume "V" and a second position in the liquid volume "V", when there is a difference in the concentration of one or more (preferably each) of the molecules M1, M2 in the mixture "M", a concentration difference ΔM, such as a gradient, is formed. Preferably, along at least one spatial coordinate "X", the second position is at a certain distance from the first position. For example, at the first position there is a corresponding first concentration of (each of) the different molecules M1, M2, and at the second position there is a corresponding second concentration of (each of) the different molecules M1, M2, wherein the corresponding second concentration is lower than the corresponding first concentration. Typically, each molecule may have a corresponding concentration at the first position (typically different concentrations for each molecule). Preferably, each of the different molecules M1, M2 is substantially absent at the second position to provide a strong contrast, such as a change in absorption. It is understood that the different molecules M1, M2 may (on average) diffuse through the liquid volume "V" according to the direction of the concentration gradient or concentration difference ΔM and / or according to their corresponding diffusion characteristics D1, D2 along the spatial coordinate "X". Thus, different molecules M1 , M2 may diffuse into (or out of) the field of view of the measurement location "P" at different times T1 , T2 depending on their respective diffusion properties D1 , D2.

[0049] In one embodiment, for example, Figure 2A As shown, the spectrum measured at the first time T1 substantially represents the spectrum characteristic graph α1 of the first molecule M1 having the highest diffusion coefficient D1 (thus, the arrival time τ1 at a certain distance from the initial position is the shortest). In another or further embodiment, Figure 2B As shown, the spectrum measured at the second time T2 basically represents a linear combination, such as the sum, of the spectral characteristic graph α1 of the first molecule M1 and the spectral characteristic graph α2 of the second molecule M2. Figure 2B The obtained spectral characteristic graphs α1, α2 and the final spectral characteristic graph αf (e.g., the sum) are shown in FIG. Figure 2C In FIG. 1 , a two-dimensional representation is shown, with the infrared frequency on the horizontal axis and the value of the diffusion coefficient obtained by analyzing the time-dependent absorption intensity at each frequency on the vertical axis. This allows the corresponding arrival times of different molecules M1, M2 to be easily shown, which can be used to determine the corresponding diffusion characteristics D1, D2, for example in combination with the known distance Xp between the measurement position "P" and the origin. Of course, these figures only represent examples that help the principle, where it should be understood that the present method is similarly applicable to any combination of different spectral feature maps α1, α2 and time-dependent evolutions τ1, τ2 that can be measured.

[0050] In some embodiments, for example, Figure 1A As shown, the concentration difference ΔM in the liquid volume "V" is provided by providing a first concentration of the mixture "M" in the first subvolume Vm. In one embodiment, the first subvolume Vm is fluidically connected to a second subvolume Vs comprising a liquid solvent "S" (suitable for dissolving different molecules M1, M2). In another or further embodiment, the liquid solvent "S" in the second subvolume Vs (at least initially) does not provide the mixture "M" and / or provides the mixture "M" at a second concentration that is lower than the first concentration of the mixture "M" in the first subvolume Vm. For example, the second concentration is at least two times, three times, five times, ten times, twenty times, fifty times, one hundred times, one thousand times or more lower than the first concentration.

[0051] In some embodiments, the first subvolume Vm comprises a mixture "M" dissolved in a liquid solvent. Preferably, the liquid solvent in the first subvolume Vm is the same or similar to the solvent in the second subvolume Vs. Alternatively, different solvents, such as (completely) miscible liquids, may be used. In principle, the mixture "M" may also start as an initial solid mixture, e.g. introduced by mixing into a liquid solvent; or start as a gas mixture, e.g. by solvent bubbling (not shown). In this case, the mixture is preferably introduced into the (liquid) first subvolume Vm and then connected to the second subvolume Vs. Alternatively or additionally, in order to initially provide the mixture "M" already dissolved in the liquid solvent, the mixture "M" may also be introduced into the liquid solvent while measuring, e.g. as Fig. 12A When the mixture "M" is introduced into the liquid solvent, it is preferably introduced relatively gently and / or at a distance from the measurement location, e.g. to avoid turbulence that could affect the diffusion process. On the other hand, it may be sufficient if there are at least some differences in the arrival times of the different molecules M1, M2 at the measurement location "P", e.g. due to their different diffusion properties D1, D2.

[0052] In some embodiments, the second subvolume Vs is fluidly connected to the first subvolume Vm via a liquid interface "I" in a direction Y perpendicular to the spatial coordinate "X". Other or further embodiments include measuring the time-dependent spectrum α(t,v) of a light beam irradiating a measurement position "P" positioned so that (only or mainly) a portion of the second subvolume Vs is exposed, while the first subvolume Vm is fluidly connected to the second subvolume Vs (e.g., sharing a liquid interface) so that different molecules M1, M2 diffuse into the field of view of the measurement position "P" at different times T1, T2 (depending on the respective diffusion characteristics D1, D2 of the different molecules M1, M2).

[0053] In some embodiments, initially, for each of the different molecules M1, M2 to be analyzed in the mixture "M", the first subvolume Vm located on one side of the liquid interface "I" has substantially a corresponding uniform concentration. In other or further embodiments, initially, the second subvolume Vs located on the other side of the liquid interface "I" does not substantially contain the different molecules M1, M2 to be analyzed in the mixture "M". Of course, it should be understood that, as used herein, references to the different molecules M1, M2 refer to the molecules of the mixture "M" to be analyzed, while the second subvolume Vs may contain the same solvent molecules, which may be considered as the background of the measured time-dependent spectrum α(t,v).

[0054] Preferably, the liquid interface "I" provides a relatively high concentration gradient, such as a sudden drop in concentration. For example, the concentration of different molecules M1, M2 to be analyzed in the mixture "M" (i.e. excluding the solvent) may change in a limited boundary area, from a (average) uniform concentration of more than 90% in the first subvolume Vm to a concentration of less than 10% in the adjacent second subvolume Vs, for example, in which the corresponding molecules to be analyzed are not present). For example, the boundary area across the initial interface I (along the direction of the spatial coordinate "X") may be less than 2 mm, less than 1 mm, less than 100 μm or even less than 10 μm. The more drastic the drop in concentration and / or the smaller the boundary area, the more clearly the starting position of the mixture (e.g., relative to the measurement position "P") is defined. For example, this clearly defined distance may help to distinguish molecules more clearly and / or to calculate the corresponding diffusion constants more accurately. It may also be helpful to select a measurement position "P" close to the initial interface "I", which may speed up the measurement process.

[0055] In some embodiments, the light "L" used to measure the time-dependent spectrum α(t,v) interacts only with a measurement position "P" located in one of the different subvolumes Vm, Vs, preferably interacts with a measurement position "P" located only in the second subvolume Vs at a measurement distance Xp from the first subvolume Vm. For example, when the measurement position "P" is in the second subvolume Vs, the light L can measure the increased absorption of the different molecules M1, M2 entering the field of view of the measurement position "P" as the different molecules M1, M2 diffuse from the first subvolume Vm to the second subvolume Vs. Alternatively or additionally, when the measurement position "P" is in the first subvolume Vm, the light L can measure the reduced absorption of the different molecules M1, M2 as some of the different molecules M1, M2 diffuse out of the field of view from the first subvolume Vm and reach the second subvolume Vs. It is also conceivable to track the corresponding concentrations at multiple measurement positions, such as one or more measurement positions for each of the different subvolumes Vm, Vs. This can provide, for example, complementary and / or reference information.

[0056] In some embodiments, the measurement position "P" is located at a predetermined distance Xp from the initial interface "I" between the first subvolume Vm and the second subvolume Vs. In other or further embodiments, the measurement position "P" has a length Y along the direction of the initial interface "I" and a width ΔX transverse to the length, wherein the width DX is smaller than the length, for example, at least two, three, five, ten or more times smaller. Preferably, the measurement position "P" has a limited measurement range ΔX at least along the spatial coordinate "X". For example, the measurement range ΔX is less than one millimeter, preferably less than half a millimeter, for example as low as one hundred microns or less. In one embodiment, the measurement position "P" is determined by an elongated hole (e.g., a slit 12c) having a length ΔY transverse to (e.g., perpendicular to) the spatial coordinate "X" and having a width ΔX along the spatial coordinate "X" to determine (e.g., limit) the measurement range ΔX. Preferably, the length ΔY of the hole is greater than the width ΔX. Most preferably, the length ΔY is arranged parallel to the liquid interface "I". Advantageously, this may allow more light to pass through the aperture to improve signal strength whilst maintaining a well-defined position Xp.Alternatively or additionally, measurements along the length of the aperture may be combined and / or averaged.

[0057] Alternatively, or in addition to using an aperture, it is also conceivable to use a focused beam (e.g. Figure 5B ). In one embodiment, the light beam "L" is focused at least in the direction of the spatial coordinate "X". In another or further embodiment, the light beam "L0" is focused in two directions X, Y. In some embodiments, the focused light beam is scanned along different measurement positions. In one embodiment, the light beam is scanned in a direction "Y" perpendicular to the spatial coordinate "X". For example, different measurements of substantially the same distance Xp from the initial liquid interface "I" can be measured and combined (e.g., averaged). In another or further embodiment, the light beam is scanned in the direction of the spatial coordinate "X". For example, this can produce additional information about the progress of diffusion of different molecules along this direction. Combinations of "X" and "Y" scans are also possible, for example, as Fig. 7A and Figure 7B Two-dimensional scanning is shown. A focused beam can also be combined with a narrow aperture, for example to ensure position over a range ΔX. For example, the focused beam can be static or scanned along the length ΔY of the aperture.

[0058] In principle, the initial concentration difference ΔM of the molecules M1, M2 between the different subvolumes Vm, Vs can be formed in various ways. In one embodiment, the initial concentration difference ΔM can be formed simply by introducing the mixture "M" at one location in the liquid volume "V" and measuring it at another location in the liquid volume "V". For example, Fig. 12AHowever, it may be difficult to fully control the process, for example, it may be difficult to determine the diffusion distances traveled by different molecules M1, M2 from the location where the molecules are introduced. In another or further embodiment, for example Fig.11A and Fig. 11B As shown, an initial concentration difference ΔM across the liquid interface "I" can be formed by initially providing different liquids separated by a barrier (e.g., a thin sheet) and removing the barrier at the beginning of the measurement. However, it may be difficult to remove such a barrier without introducing turbulence into the liquid volume "V", which may affect diffusion. These and other disadvantages can be alleviated by establishing an initial concentration difference ΔM according to a preferred embodiment disclosed below.

[0059] FIG. 3A to FIG. 3C The use of a set of parallel flows Fm, Fs to provide an initial concentration difference ΔM between different subvolumes Vm, Vs is shown. In some embodiments, the first subvolume Vm and the second subvolume Vs as described herein are provided by generating a set (e.g., a pair) of parallel laminar flows Fm, Fs, comprising a first flow Fm containing a mixture "M" of different molecules and an adjacent second flow Fs containing a liquid solvent "S" (without the molecules, or at least a more diluted solution), with a flow interface "If" between at least a portion of the first flow Fm and the second flow Fs. Other or further embodiments include stagnating the flows Fm, Fs to provide a (static) first subvolume Vm formed by the stagnant first flow Fm and a (static) second subvolume Vs formed by the stagnant second flow Fs. Preferably, the first subvolume Vm is fluidly connected to the second subvolume Vs via a liquid interface "I" formed by a previous flow interface "If". Advantageously, by providing a laminar flow, the flow interface "If" can be relatively clearly defined and used as an origin in modeling. In principle, more than two parallel flows can also be used, for example three parallel flows, wherein the same or different mixtures are provided in the outer flows and the solvent is provided in the central flow without the mixture. Alternatively, different mixtures can be provided in two or more adjacent flows and the solvent is provided in another flow, for example an outer flow. Preferably, the parallel flows are arranged in a plane, although in principle more complex flows can also be envisaged.

[0060] FIG. 4A to FIG. 4C Aspects of a sample cell 10 configured to provide parallel flow in an elongated flow channel 12c are shown. Specifically, Figure 4A Shows the formation Figure 4B possible configurations of the elements of the sample pool 10 in; and Figure 4C Shown with Figure 4B. A photograph of an actual sample cell 10 similar to the illustration of the drawing. In some embodiments, for example, as shown in the figure, different subvolumes Vm, Vs are provided in the sample cell 10 (e.g., a flow cell). In one embodiment, the sample cell 10 includes an elongated flow channel 12c. In another or further embodiment, the sample cell 10 includes a first input port 11m, which is used to introduce a mixture "M" having different molecules M1, M2 to be analyzed as a first flow Fm into the elongated flow channel 12c. In another or further embodiment, the sample cell 10 includes a second input port 11s, which is used to introduce a liquid solvent "S" as a second flow Fs into the same elongated flow channel 12c. In another or further embodiment, the sample cell 10 includes at least one outlet port 11e, which is used to allow the mixture "M" and / or the liquid solvent "S" to leave the elongated flow channel 12c. Preferably, the input port 11m, the input port 11s is connected to the slender flow channel 12c via corresponding converging channels 12m, the converging channels 12s, and the converging channels 12m, the converging channels 12s are arranged adjacent to each other in a direction (X) transverse to the length direction (Y) of the slender flow channel 12c to generate a set of parallel laminar flows Fm, Fs including a first flow Fm and a second flow Fs along the length of the slender flow channel 12c toward at least one outlet port 11e.

[0061] In some embodiments, for example, Figure 4A As shown, the sample pool 10 includes a front cover 11, a rear cover 13 and a spacer 12. In one embodiment, the spacer 12 forms a flow structure 12m, a flow structure 12s, and a flow structure 12c. In another or further embodiment, an input port 11m, an input port 11s and / or an outlet port 11e are formed in the front cover 11. Of course, one or more of these ports may also be formed in the rear cover 12 (not shown), and / or formed by a side opening in the spacer 12 (not shown). As an alternative to the modular structure shown, one or more of the elements shown may also be integrated. Preferably, the front cover 11 and / or the rear cover 13 are formed by corresponding windows, which allow transmission of light for measuring the interior of the sample. For example, when the sample is measured by transmitting an infrared beam through the sample, the front cover 11 and the rear cover 13 may be formed by a calcium fluoride window or a similar material that is transparent in the infrared wavelength range. It is also conceivable that, for example, in a reflection-based setting, only one cover is transparent.

[0062] In some embodiments, an adjustable hole is provided that allows the position (X and / or Y) and / or size (ΔX and / or ΔY) of the measurement position "P" to be adjusted. In one embodiment, for example, as shown, the sample cell 10 includes or is connected to a plate 14 having a slit 14s, which provides a hole that determines the measurement position "P" in the elongated flow channel 12c. In one embodiment, the plate 14 is an integrated part of the sample cell 10. In another embodiment, the plate 14 is independent of the sample cell 10. Preferably, the independent or integrated plate is adjustable, for example allowing the measurement position "P" to be adjusted relative to the elongated flow channel 12c. Alternatively, in addition to using a single plate to form a slit, it is also conceivable to use two plates, for example to form a slit between the edges of these plates, or partially overlapping slits (not shown). For example, this allows the width and / or length of the slit to be adjusted as well.

[0063] Figure 5A Other aspects of the sample cell 10 are shown. In some embodiments, an opaque structure (e.g., a plate) including an elongated hole (e.g., a slit) is provided, which is arranged to expose a measurement position "P" corresponding to one of the parallel flows in the elongated flow channel 12c, while blocking light from passing anywhere else. In some embodiments, the length 12l of the flow channel 12c is greater than its width 12w, such as at least two times, three times, five times, up to ten times or more. In other or further embodiments, the thickness 12d of the elongated flow channel 12c (e.g., as Figure 4A As shown, in a direction perpendicular to the length 12l and the width 12w), it is relatively small, for example, less than 5 mm, preferably less than 3 mm, more preferably less than 2 mm, and most preferably less than 1 mm. For example, the channel thickness 12 is relatively small compared to the width 12w and / or the length 12l of the flow channel 12c, for example, at least 2 times, 3 times, 5 times, up to 10 times or more. The inventors have found that by keeping the flow channel relatively thin, the flow can be better controlled, for example, to maintain laminar flow and / or avoid turbulence, which otherwise may affect the diffusion measurement.

[0064] Preferably, the elongated hole has a length ΔY arranged along the length 121 of the elongated flow channel 12c and a width ΔX (perpendicular to the length) that is less than the half width 12hw (i.e., half of the width 12w) of the elongated flow channel 12c, for example, the width ΔX is at least two, three, five or more times smaller than the half width 12hw. For example, the measurement position "P" can be arranged to expose a portion of the lower half and / or a portion of the upper half of the channel. Of course, this can depend on the orientation of the sample cell 10 and / or the specific connection of the corresponding liquid supply to the input ports 11m, 11s. Most preferably, the slit 14s for allowing light to enter and / or leave the elongated flow channel 12c is arranged to expose less than half of the elongated flow channel 12c on one side corresponding to the flow of the liquid solvent "S" (e.g., on the side of the corresponding input port 11s). Therefore, light can be blocked from passing through other parts of the channel. For example, the light beam used to measure the spectrum is configured to pass only through a relatively large portion of the flow channel that has a liquid solvent or a pure liquid solvent at least initially.

[0065] Figure 5B A system 100 is shown for spectroscopic analysis of a mixture "M" of different molecules M1, M2, for example, as described herein. In some embodiments, the system includes a container, such as a sample cell 10, which is configured to provide a liquid volume "V" having a mixture "M", the mixture "M" having an initial concentration difference ΔM between different sub-volumes Vm, Vs of the liquid volume "V", the different sub-volumes Vm, Vs being fluidically connected and arranged at different positions along a spatial coordinate "X". In other or further embodiments, the system includes a measuring device, such as a spectrometer 30, which is configured to measure a time-dependent spectrum α(t,v) of light L interacting with the liquid volume "V" at a measurement position "P" located in (at least) one of the different sub-volumes Vm, Vs while the concentration difference ΔM is at least partially balanced by diffusion of the different molecules M1, M2 between the different sub-volumes Vm, Vs along the spatial coordinate "X".

[0066] In some embodiments, the system includes a processing device 50 configured to receive and / or process a time-correlated spectrum α(t, v). For example, the spectrum may be processed while and / or after receiving the spectrum from a spectrometer and / or a measuring device. In one embodiment, the processing device includes a general purpose processor. In another or further embodiment, the processing device includes a dedicated circuit to perform any method as described herein. In some embodiments, the system includes a clock, which may be part of the measuring and / or processing device. Typically, the processing device 50 may include and / or access a (non-transitory) computer-readable medium storing instructions that, when executed, cause the processing device to perform an operation as described herein, for example, processing the time-correlated spectrum α(t, v). In one embodiment, the time-correlated spectrum α(t, v) is processed to determine corresponding different spectral feature graphs α1, α2 of corresponding different molecules M1, M2, and / or to determine corresponding different diffusion characteristics D1, D2 of different molecules M1, M2 based on different time-correlated evolutions τ1, τ2 of each spectral feature graph α1, α2 in the time-correlated spectrum α(t, v).

[0067] Alternatively, or in addition to determining the spectral signatures α1, α2 and / or the diffusion characteristics D1, D2, other or relevant characteristics of the different molecules M1, M2 may be determined, such as arrival time, molecular mass, relative size, molecular type, etc. In principle, various types of sample cells may be used to establish the initial concentration difference ΔM, for example, as described with reference to any one or more of FIGS. 1, 3, 11, 12. In a preferred embodiment, as described with reference to FIG. 4A to FIG. 4C , Figure 5A , Fig. 6A , Figure 6B , Fig. 7A The sample cell 10 is used as described in any one or more of the foregoing.

[0068] In some embodiments, the system includes a pumping arrangement 40 having a pumping device 42 configured to receive a mixture "M" and a liquid solvent "S" and to generate a set of parallel laminar flows Fm, Fs in the sample chamber 10. For example, the pumping device 42 includes one or more peristaltic pumps or other types of pumps to generate at least two separate flows. Of course, separate pumping devices may also be used, for example, one pumping device for each flow. In one embodiment, the system (e.g., a processing device 50 and / or other controller (not shown)) is configured to control the pumping device 42 to stop (or significantly slow down) the flow when the time-dependent spectrum α(t, v) is measured. For example, the flow is stagnant at a starting time T0. Thus, the system can measure a time-dependent spectrum α(t, v) including an indication of the time (t) that has elapsed since the starting time T0. For example, an indication of the time (t) can be recorded with each spectrum.

[0069] In some embodiments, the pumping configuration 40 includes a first reservoir 41m for containing the mixture "M" and / or a second reservoir 41s for containing the liquid solvent "S". In one embodiment, the reservoirs 41m, 41s are connected to the sample cell 10 via a pumping device 42 to supply the mixture "M" and the liquid solvent "S" to the first input port 11m and the second input port 11s, respectively. In another or further embodiment, the pumping configuration 40 includes a waste reservoir 43, which is connected to at least one outlet port 11e and is configured to receive the mixture "M" and / or the liquid solvent "S" after passing through the sample cell 10.

[0070] In some embodiments, the spectrometer 30 includes a spectral resolving element 31, such as a grating, a prism, or a Fourier-transform (infrared) spectrometer. In other or further embodiments, the spectrometer 30 includes a light sensor 32, such as a pixel array, which is configured to measure the light intensity of infrared light or any other light. In one embodiment, for example, as shown in the figure, the spectral resolving element 31 is configured to receive a light beam Lt that has interacted with the liquid volume "V" in the sample cell 10, and send a spectrally resolved light beam Ls to the light sensor 32. Typically, the spectrometer can use a slit that is used to spatially shape the light (e.g., a pixel) imaged onto the light sensor 32. The sensing configuration 30 can also include other or further optical components to shape and / or redirect the light beam, and / or filter the light. For example, the sensing configuration 30 can include a collimating lens 33. Alternatively or additionally, a curved mirror can also be used.

[0071] In some embodiments, the spectrometer is configured to image the elongated flow channel 12c or a portion thereof onto the light sensor 32. In other or further embodiments, the spectrometer is configured to image the slit 14s onto the light sensor 32. In one embodiment, the spectrometer includes an optical system (not shown) having an object plane coinciding with an aperture (e.g., slit 14s) for determining the measurement position "P" and an imaging plane coinciding with the light sensor 32. For example, a spectrally resolved image of the slit 14s is imaged onto the sensor array. Alternatively or additionally, a single point (e.g., the focus of the light beam "L0" passing through the slit) can be spectrally resolved and imaged onto the light sensor 32. It is also conceivable to use another slit (not shown), for example, after refocusing all light passing through the slit 14s adjacent to the elongated flow channel 12c.

[0072] In some embodiments, the spectrometer 30 includes or is coupled to the lighting device 20. In other or further embodiments, the lighting device 20 includes a light source 21, such as an infrared light source. For example, the light source includes a lamp or a laser. In one embodiment, the light source 21 is integrated as part of the measurement system. In another embodiment, the light source is separate. In one embodiment, for example, as shown, the source light L0 from the light source 21 is focused at a measurement position "P" in the sample pool. For example, the light can be focused onto a slit, or the slit can be omitted. In another embodiment, an unfocused light beam is used in conjunction with a finite aperture. Therefore, it should be understood that the measurement position "P" can be determined by the position and / or range of a physical aperture (e.g., a slit that overlaps a portion of the sample), the position and / or size of the light beam (e.g., focus), and / or a combination thereof.

[0073] In some embodiments, the system includes a positioning device 15, which is configured to determine the position and / or size of the measurement position "P" relative to different sub-volumes Vm, Vs in the sample cell 10. In one embodiment, the positioning device 15 is configured to adjust the position and / or size of the physical hole overlapping with a portion of the liquid volume "V" in the sample cell 10. In another or further embodiment, the positioning device 15 is configured to adjust the position and / or size of the light beam interacting with a portion of the liquid volume "V" in the sample cell 10. In another or further embodiment, the positioning device 15 is configured to adjust the position of the sample cell 10, for example relative to the physical hole and / or relative to the position and / or focus of the light beam "L" 0. Advantageously, by moving only the sample cell, the rest of the optical system (e.g., focus position, hole position, etc.) can remain stationary. Of course, it is also possible to move a combination of one or more components.

[0074] Fig. 6A and Figure 6B A photograph of a positioning device 15 configured to adjust a measurement position "P" in a sample chamber 10 is shown. In some embodiments, the system includes one or more plates 14 that form a slit 14s that overlaps a portion of the sample (e.g., an elongated flow channel 12c). In other or further embodiments, the system includes a positioning device 15 that is configured to adjust the position and / or size of the slit 14s relative to different subvolumes Vm, Vs in the sample (e.g., relative to the elongated flow channel 12c).

[0075] Fig. 7AThe infrared microscope image of channel 12c is shown when a solution containing D2O-PEG 2wt%-acetone 4wt%-L-alanyl-L-alanine (dialanine) 60mg / ml is injected as a mixture "M" and a liquid solution "S" containing D2O-PEG 2wt%. The color bar indicates acetone at 1699cm -1 The absorption intensity at the absorption frequency.

[0076] Figure 7B and Figure 7C Infrared microscope images at different times T after the flow has stopped are shown. Figure 7B IR-microscope images of the channel cross section taken at different times after stopping the injection are shown, representing the characteristic absorption frequency of acetone at 1699 cm -1 The absorption intensity at . Figure 7C shows a similar absorption frequency of 1595 cm-1 but at the characteristic absorption frequency of L-alanyl-L-alanine -1 The image at (also see Fig. 9B ). The intensity of absorption is proportional to the concentration of the molecule. IR images are taken by scanning the beam in the XY plane with a fixed focus using an infrared microscope.

[0077] Fig. 8A The time-dependent infrared absorption spectra of a mixture of different molecules M1, M2, M3 diffused into the field of view of the measurement location are shown. As shown, different molecules have different absorption peaks that evolve (here increase) at different time rates τ1, τ2, τ3. Figure 8B The corresponding diffusion ordered spectrogram is shown. The spectrogram essentially represents a two-dimensional contour plot of the absorption intensity as a function of frequency "ν" and the diffusion coefficient. As shown, the time dependence can be transformed to determine the corresponding diffusion coefficient "D". For example, the rescaling can be performed based at least in part on the distance between the initial position of the mixture (e.g., the initial liquid interface) and the measurement position (e.g., the focus of the slit and / or light beam). In this particular measurement, the mixture used contained: M1 = acetone (4wt%); M2 = glucuronic acid (40mg / ml); M3 = bovine serum albumin (60mg / ml). The measurements were performed using a Fourier transform infrared (FTIR) spectrometer in transmission mode. Of course, it should be understood that the concept is not limited to a particular mixture or measurement setup.

[0078] Fig.9A and Fig. 9B Additional spectra are shown. Fig.9A Similar to Figure 8B ,but Fig.9Ais obtained from spatially resolved data recorded using an IR microscope. In this case, the absorption data are obtained as a function of time and position α(t,X), where X is the position in the direction perpendicular to the channel. Fig. 9B A spectrum diagram illustrating the diffusion coefficient "D" of a mixture containing L-alanyl-L-alanine / acetone as a function of frequency is shown. Figure 7B The 2D absorption data obtained using IR microscopy are shown averaged to extract the time-dependent absorption curve. Figure 8B and Fig. 9B The fact that the diffusion coefficients shown in are similar indicates that FTIR spectroscopy can be used to determine the diffusion coefficient from the time-dependent absorption intensity just like FTIR microscopy. These figures also illustrate that the diffusion constant is related to the molecular weight (here expressed in (kilo)Daltons).

[0079] Fig.10 Two-dimensional infrared pump-probe spectra recorded after stopping injection of a mixture "M" containing D2O-PEG 2wt%-acetone 4%-bovine serum albumin ("BSA") 50 mg / ml in inlet 11m and stopping injection of a solution "S" containing D2O-PEG 2wt% in inlet 11s are shown. Each scan from T=1 to T=43 reports the average 2D signal for 15 minutes. Note that the first scan depicted at T=1 has a different scale compared to the other scans at T=12, 20, and 43. The spectral gap corresponding to the acetone absorption peak (ν=0→1) first appears at 1700 cm -1 The spectral gap corresponding to the BSA absorption peak appears at 1660 cm -1 At a later scan (T≥20). In this experiment, the 2D absorption spectrum was measured by using a slit at a single position at the end of the channel in a homemade 2DIR setup described in the literature Huerta-Viga, A.; Shaw, DJ; Woutersen, S. pH Dependence of the Conformation of Small Peptides Investigated with Two-Dimensional Vibrational Spectroscopy. Journal of Physical Chemistry B 2010, 114, 15212–15220. The waiting time between the pump and the probe was 1 ps.

[0080] As will be appreciated, two-dimensional and / or pump-probe spectra can reveal additional information beyond linear spectra. For example, the corresponding anharmonicities δ1, δ2 (the difference in transitions between ν=0→1 and ν=1→2, represented by "-" and "+", respectively) can be determined for each molecule M1, M2 and used for further identification and / or structural analysis. It also illustrates that the present method and system are not limited to measuring linear spectra. For example, Raman spectroscopy and the like can also be used to apply to the same or similar features. It should also be understood that the spectral feature maps α1, α2 as described herein are not limited to one-dimensional spectra, but can include multi-dimensional spectra of each molecule, for example, a series of 2D spectra that evolve over time. Other or additional data structures and corresponding modeling can also be envisioned. A common feature is, for example, tracking different time-dependent evolutions of different spectral features of different molecules.

[0081] FIG. 11A to FIG. 11C A liquid volume "V" of a mixture "M" having an initial concentration difference ΔM is shown provided by removing a barrier 19 between different subvolumes Vm, Vs. In some embodiments, a container (e.g., a sample cell) includes a liquid volume "V" having a barrier 19 that initially separates a first subvolume Vm from a second subvolume Vs. For example, the first subvolume Vm consists essentially of the mixture "M" and the second subvolume Vs consists essentially of a liquid solvent "S". In one embodiment, the barrier 19 is removed at the beginning of the measurement. For example, the barrier 19 is pulled out from between the subvolumes Vm, Vs, which allows different molecules M1, M2 to begin to diffuse according to the concentration difference ΔM between the first subvolume Vm and the second subvolume Vs. Preferably, the barrier 19 is relatively thin, such as much less than 1 mm, so that minimal turbulence is generated when the barrier 19 is pulled out of the liquid volume "V". Most preferably, the barrier 19 having a planar shape is pulled out along an in-plane direction to minimize the displacement of liquid in the process.

[0082] In one embodiment, for example, as shown, at least one measurement position Ps is located in the second subvolume Vs, i.e., a volume having a relatively low concentration of the mixture "M" and / or having no concentration of the mixture "M" initially (e.g., pure liquid solvent "S"). In another or further embodiment, at least one measurement position Pm is located in the first subvolume Vm, i.e., a volume having a relatively high or initial concentration of the mixture "M". In some embodiments, the time-correlated spectrum of light interacting with the liquid volume "V" is measured (simultaneously or continuously) at two or more measurement positions Ps, Pm. In other or further embodiments, the concentration difference ΔM is measured using a first measurement position Pm in the first subvolume Vm and a second measurement position Ps in the second subvolume Vs. In one embodiment, the time-correlated spectrum α(t,v) of the light L interacting with the liquid volume "V" is measured based on the measured concentration differences ΔM between different positions. For example, the first measurement position "Pm" in the first subvolume Vm can be used as a reference measurement and / or for improving the measurement contrast of the time-evolving concentration differences ΔM between different subvolumes. For example, this can help mitigate other time-dependent changes, such as temperature fluctuations of the liquid volume "V". It should be understood that the advantageous features regarding measurements at two or more locations (e.g., as reference measurements) are not limited to the specific embodiment of Figure 11, but can be similarly applied to any embodiment as described herein, including Figures 1 and 2. For example, two or more slits and / or light beams can be used at different locations. More than two measurement locations can also be used, for example, a set of three, four or more locations at different values ​​of the spatial coordinate "X" (along the concentration gradient / concentration difference ΔM, not necessarily at the same Y value). These and other features can also be applied to the embodiment of Figure 12.

[0083] FIG. 12A to FIG. 12C It is shown that a liquid volume "V" with a mixture "M" is provided, which mixture "M" has an initial concentration difference ΔM between a first subvolume Vm to which the mixture "M" is added and a second subvolume Vs including a measurement position "P". In principle, as long as there is at least some effect of the different molecules M1, M2 diffusing into (or out of) the field of view of the measurement position "P" at different times T1, T2 (depending on the respective diffusion characteristics D1, D2 of the different molecules M1, M2), the respective spectral components of the different molecules M1, M2 can be extracted based on the time-correlated spectra. For example, the mixture can be (gently) added to a part of the solvent, or vice versa, while minimizing convection (e.g. turbulence) at least in an area of ​​the second subvolume Vs close to the measurement position "P" (e.g. the measurement position "P"). For example, the measurement position "P" can be located relatively far away from where the mixture "M" is added to the liquid solvent "S". Preferably, for example, as Figure 1Aand Figure 3B As shown, the first subvolume Vm containing the mixture "M" initially has a relatively uniform concentration of the different molecules M1, M2, for example forming a well-defined initial interface "I" between the volumes, and allowing the determination of an exact distance Xp between the interface "I" and the measurement position P (e.g., a window, a slit, a focus of a light beam, etc.). However, this is not strictly necessary, as long as the diffusion characteristics D1, D2 still allow the different molecules M1, M2 to diffuse into the field of view of the measurement position "P" at different times.

[0084] In some embodiments, the time-correlated spectrum is measured by a light beam passing through a measurement area of ​​a liquid solvent "S", while a mixture "M" of different molecules M1, M2 is introduced at an insertion position, which is fluidically connected to the measurement position "P", but is remote from the measurement position "P". Preferably, the light beam does not pass through the insertion position. In other or further embodiments, the measurement of the time-correlated spectrum α(t,v) starts from the measurement position "P" without the mixture "M" and continues while the different molecules M1, M2 enter the measurement position "P" by corresponding diffusion. Of course, the measurement does not necessarily have to start at the exact time of the insertion, but can also start when the equilibrium process has already started but has not yet been completed.

[0085] Fig.13A and Fig. 13B shows that using relatively large channel thickness (e.g. Figure 4A 12d) of the experiment shown in FIG. In the experiment shown, the channel thickness was 2.2 mm. These figures show photographs of the flow patterns obtained by adding dye to the sample solution. Fig.13A In the present invention, it can be observed that the use of relatively deep channels may lead to turbulence in the flow. These turbulences may lead to premature mixing of the flow, which may negatively affect the ability to produce a clear initial liquid interface when performing measurements as described herein (e.g., IR-DOSY). To alleviate this problem, certain properties of the channel and / or the liquid may be adapted to reduce the occurrence of turbulence while still allowing the desired measurements to be performed.

[0086] Without being bound by theory, the occurrence of laminar or turbulent flow can be modeled using the Reynolds number "Re", which can be defined as Re = u·L / ν = ρ·u·L / μ, where ρ is the density of the fluid (SI units: kg / m 3 ), “u” is the flow velocity (m / s), “L” is the characteristic linear dimension (m), and “μ” is the dynamic viscosity of the fluid (Pa·s or N·s / m 2 or kg / (m·s)), “ν” is the kinematic viscosity of the fluid (m 2 / s). Generally, laminar flow occurs when the Reynolds number is relatively low, and turbulent flow tends to occur when the Reynolds number is relatively high. Therefore, in some embodiments, one or more measures can be taken to avoid the occurrence of turbulent flow.

[0087] In some embodiments, methods and systems for providing subvolumes by generating a pair of parallel laminar flows (e.g., as described with reference to any one of Figures 3 to 5) include using a flow with a relatively low flow rate. In one embodiment, to avoid turbulence, the flow rate is preferably less than five millimeters per second, more preferably less than one millimeter per second, and most preferably less than half a millimeter per second. In another or further embodiment, it may be desirable to speed up the process and / or avoid a certain minimum flow rate at which significant diffusion of smaller solutes has occurred during liquid injection. Therefore, the flow rate is preferably at least 10 microns per second, more preferably at least 25 microns per second, more preferably at least 50 microns per second, and most preferably at least 100 microns per second.

[0088] In other or further embodiments, methods and systems for providing subvolumes by generating a pair of parallel laminar flows include using a relatively low channel thickness. In one embodiment, to avoid turbulence, the channel thickness is preferably less than 1 mm, more preferably less than 0.5 mm, and most preferably less than 0.1 mm. In another or further embodiment, the channel thickness is preferably high enough to obtain a measurable absorption of light passing through the channel. This may depend on the absorption coefficient and / or concentration of the solute and / or solvent. Typically, the channel thickness is at least fifty microns, preferably at least one hundred microns, more preferably at least two hundred microns, for example up to five hundred microns or more.

[0089] In other or further embodiments, methods and systems for providing subvolumes by generating a pair of parallel laminar flows include using flows with relatively high viscosities ("ν" and / or "μ"). In one embodiment, to avoid turbulence, the viscosity of at least one fluid, preferably both fluids, forming the parallel flows is at least 0.11 mPa·s, more preferably at least 0.5 mPa·s, most preferably at least 1 mPa·s. In another or further embodiment, the viscosity should not be so high as to hinder flow and / or hinder diffusion. Therefore, the viscosity is preferably less than 20 mPa·s, more preferably less than 10 mPa·s, most preferably less than 5 mPa·s, for example less than 2 mPa·s, or less.

[0090] Among the possible measures that can be taken, the inventors have found that turbulence may be most easily avoided by using suitable additives in one or both of the flowing liquids. For example, this may affect viscosity. In some embodiments, additive molecules are added to at least one of the sample solution (mixture M) and the solvent solution (liquid solvent S), preferably both. Preferably, the additive molecule is an inert polymer. For example, a polymer that dissolves well in the solvent and has minimal interaction with the solute under study (e.g., does not react with the solute under study) is selected. Preferably, the concentration of the additive is relatively low, such as less than 5wt% of the solvent, more preferably less than 3wt%, such as between 1wt% and 2wt%, or less. For example, for aqueous solutions, polyethylene glycol (PEG) has been found to be particularly suitable, but other additives are also possible. In a preferred embodiment, a certain amount of additive is added to at least one fluid, wherein the type and concentration of the additive are selected so that the viscosity increases (in the absence of additives) by at least two times, preferably at least five times, more preferably at least ten times, up to twenty times, or more. For example, Fig. 13B Shown with Fig.13A The same experiment, except the additive polyethylene glycol was added at a concentration of 2.5 g / L. As can be observed, the additive greatly improved the stratification of the flow pattern.

[0091] In interpreting the appended claims, it should be understood that the word "comprising" does not exclude the presence of other elements or acts than those listed in a given claim; the word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements; any reference signs in the claims do not limit their scope; several "devices" may be represented by the same or different items or implemented structures or functions; unless otherwise specifically stated, any disclosed device or part thereof may be combined together or divided into further parts.

Claims

1. A method for spectroscopic analysis of a mixture (M) of different molecules (M1, M2), the method comprising: providing a liquid volume (V) having said mixture (M), said mixture (M) having an initial concentration difference (ΔM) between different sub-volumes (Vm, Vs) of said liquid volume (V), said different sub-volumes (Vm, Vs) being fluidically interconnected and arranged at different positions along a spatial coordinate (X); and measuring a time-dependent spectrum (α[t,v]) of light (L) interacting with the liquid volume (V) at a measurement position (P) located in one of the different subvolumes (Vm, Vs) while the concentration difference (ΔM) is at least partially balanced by diffusion of the different molecules (M1, M2) between the different subvolumes (Vm, Vs) along the spatial coordinate (X); Wherein, the time-correlated spectrum (α[t,ν]) includes corresponding different spectral characteristic graphs (α1, α2) of the corresponding different molecules (M1, M2), and each of the spectral characteristic graphs (α1, α2) has different time-correlated evolutions (τ1, τ2) in the time-correlated spectrum (α[t,ν]) caused by corresponding different diffusion characteristics (D1, D2) of the different molecules (M1, M2).

2. The method according to the preceding claim, wherein: The concentration difference (ΔM) in the liquid volume (V) is provided by supplying a first concentration of the mixture (M) having the different molecules (M1, M2) to be analyzed in a first subvolume (Vm), wherein the first subvolume (Vm) is fluidically connected to a second subvolume (Vs), the second subvolume (Vs) containing a liquid solvent (S) suitable for dissolving the different molecules (M1, M2) to be analyzed, wherein the liquid solvent (S) in the second subvolume (Vs) does not have the mixture (M) at least initially.

3. The method according to the preceding claim, wherein: The first subvolume (Vm) comprises the mixture (M) dissolved in the same liquid solvent (S) forming the second subvolume (Vs).

4. The method according to the preceding claim, in, The second subvolume (Vs) is adjacent to the first subvolume (Vm) and is integrally connected via a liquid interface (I) extending along a direction (Y) perpendicular to the spatial coordinate (X) to form a continuous liquid volume (V); Therein, the light beam (L) used to measure the time-correlated spectrum (α[t,ν]) interacts only with a measurement position (P) located only in the second subvolume (Vs) at a measurement distance (Xp) from the initial liquid interface (I) between the first subvolume (Vm) and the second subvolume (Vs).

5. A method according to the preceding claim, wherein: At the beginning, The first subvolume (Vm) located on one side of the liquid interface (I) has a substantially uniform concentration for each of the different molecules (M1, M2) to be analyzed in the mixture (M); and The second subvolume (Vs) situated on the other side of the liquid interface (I) is substantially free of the different molecules (M1, M2) to be analyzed in the mixture (M).

6. A method according to the preceding claim, wherein: The first subvolume (Vm) and the second subvolume (Vs) are provided by: generating a pair of parallel laminar flows (Fm, Fs), comprising a first flow (Fm) comprising the mixture (M) of different molecules and an adjacent second flow (Fs), wherein the first flow (Fm) comprises the mixture (M) of different molecules, and the second flow (Fs) comprises the liquid solvent (S), wherein a flow interface (If) exists between at least a portion of the first flow (Fm) and the second flow (Fs); and The flows (Fm, Fs) are stagnated to provide a first subvolume (Vm) formed by the stagnant first flow (Fm) and a second subvolume (Vs) formed by the stagnant second flow (Fs), wherein the first subvolume (Vm) is fluidically connected to the second subvolume (Vs) via a liquid interface (I) formed by the previous flow interface (If).

7. A method according to the preceding claim, wherein: The pair of parallel laminar flows (Fm, Fs) are generated in an elongated flow channel (12c), wherein the measurement position (P) has a limited measurement range (ΔX) along the spatial coordinate (X) that is less than half of the width (12w) of the elongated flow channel (12c).

8. A method according to any one of the preceding claims, wherein: The measurement position (P) is determined by an elongated slit (14s) having a length (ΔY) perpendicular to the spatial coordinate (X) and having a width (ΔX) along the spatial coordinate (X) to limit the measurement range (ΔX).

9. A method according to any one of the preceding claims, wherein: The measurement position (P) is determined by the shape of the light beam focused in one of the different sub-volumes (Vm, Vs) at least along the spatial coordinate (X).

10. A method according to any one of the preceding claims, wherein Based on the time-dependent spectrum (α[t,ν]), the mixture (M) is analyzed by at least one of the following: Determining the corresponding different spectral characteristic patterns (α1, α2) of the different molecules (M1, M2); determining corresponding different diffusion characteristics (D1, D2) of said different molecules (M1, M2); determining the frequency and / or amplitude of spectral features in one or more spectral feature maps (α1, α2) of the different molecules (M1, M2); determining the corresponding sizes of one or more of the different molecules (M1, M2) in the mixture (M); identifying one or more of said different molecules (M1, M2) in said mixture (M); determining one or more corresponding concentrations of said different molecules (M1, M2) in said mixture (M); The molecular structure of one or more of the different molecules (M1, M2) in the mixture (M) is determined.

11. A method according to any one of the preceding claims, wherein A first measurement position (Pm) is located in the first subvolume (Vm) having the mixture (M) and initially containing the different molecules (M1, M2) to be analyzed, and a different second measurement position (Ps) is located in the second subvolume (Vs) having the liquid solvent (S) and initially not containing the different molecules (M1, M2), wherein the time-dependent spectrum (α[t,ν]) of light interacting with the liquid volume (V) is measured at both the first and second measurement positions (Ps, Pm) to determine a concentration difference (ΔM) evolving over time between the different subvolumes (Vm, Vs).

12. A system (100) for spectroscopic analysis of a mixture (M) of different molecules (M1, M2), the system comprising: a sample cell (10) configured to provide a liquid volume (V) having the mixture (M), the mixture having an initial concentration difference (ΔM) between different subvolumes (Vm, Vs) of the liquid volume (V), the different subvolumes (Vm, Vs) being fluidically interconnected and arranged at different positions along a spatial coordinate (X); and A measuring device (20, 30) comprising a spectrometer (30) configured to measure a time-dependent spectrum (α[t, ν]) of light (L) interacting with the liquid volume (V) at a measurement position (P) located in one of the different subvolumes (Vm, Vs) while the concentration difference (ΔM) is at least partially balanced by diffusion of the different molecules (M1, M2) between the different subvolumes (Vm, Vs) along the spatial coordinate (X); and A processing device (50) having a non-transitory computer-readable medium storing instructions, which when executed cause the processing device to process the time-correlated spectrum (α[t,ν]) to determine corresponding different spectral feature graphs (α1, α2) of the corresponding different molecules (M1, M2), and determine corresponding different diffusion characteristics (D1, D2) of the different molecules (M1, M2) based on different time-correlated evolutions (τ1, τ2) of each spectral feature graph (α1, α2) in the time-correlated spectrum (α[t,ν]).

13. A system according to the preceding claim, wherein: The sample pool (10) comprises An elongated flow channel (12c); a first input port (11m) for introducing the mixture (M) with the different molecules (M1, M2) to be analyzed as a first flow (Fm) into the elongated flow channel (12c); a second input port (11s) for introducing said liquid solvent (S) as a second flow (Fs) into the same said elongated flow channel (12c), and at least one outlet port (11e) for allowing the mixture (M) and / or the liquid solvent (S) to exit the elongated flow channel (12c); The input ports (11m, 11s) are connected to the elongated flow channel (12c) via corresponding converging channels (12m, 12s), and the converging channels (12m, 12s) are arranged adjacent to each other in a direction transverse to the length of the elongated flow channel (12c) to generate a set of parallel laminar flows (Fm, Fs) including the first flow (Fm) and the second flow (Fs) along the length of the elongated flow channel (12c) toward the at least one outlet port (11e).

14. A system according to the preceding claim, comprising at least one plate (14) forming a slit (14s), the slit (14s) being arranged to expose less than half of the elongated flow channel (12c), and A positioning device (15) is configured to adjust the position and / or size of the slit (14s) relative to the different sub-volumes (Vm, Vs) in the elongated flow channel (12c).

15. A system according to the preceding claim, wherein: The system comprises a pumping arrangement (40) having a pumping device (42) configured to receive the mixture (M) and the liquid solvent (S) and to generate a set of parallel laminar flows (Fm, Fs) in the sample cell (10), wherein the system is configured is arranged to control the pumping device (42) to stop the flow at a starting time (T0) and to measure the time-dependent spectrum (α[t,ν]), The time-dependent spectrum (α[t,ν]) comprises an indication of the time (t) that has elapsed since the start time (T0).

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