Multi-dimensional online photoreaction monitoring system

Abstract

The utility model discloses a multidimensional on-line photoreaction monitoring system, including sample pump, transparent pipeline, programmable LED array and mass spectrometer, wherein the front end of transparent pipeline is connected with sample pump, the rear end is connected with mass spectrometer, the middle is snakelike bending area, the uniform light field generated by programmable LED array irradiates the snakelike bending area. A reaction sample is injected into the transparent pipeline through the sample pump at a constant flow rate, is irradiated by a uniform light field in the snake-shaped bent area to carry out photoreaction, and then is input into the mass spectrometer to carry out on-line real-time multi-reaction monitoring (MRM). The light field generated by the programmable LED array is uniform and stable, the irradiation time t and the optical power density [hv] can be modulated, the problems that an internal standard substance is needed, signals are unstable, the adjustable range is small and the dead time is long in variable flow velocity online mass spectrum monitoring are solved, the data acquisition speed is increased, and an effective solution is provided for research of a photochemical kinetic mechanism.

Description

Technical Field

[0001] The utility model relates to an online photochemical reaction monitoring system, in particular to a system for realizing online regulation of light power density and illumination time and online monitoring of mass spectrum. Background Art

[0002] Photocrosslinking (PXL) using alkyl diaziridine can provide strict distance restrictions and in-depth information on protein structure, but the mechanism is unclear, the technology is not unified, and there is a lack of standardized, controllable, and stable instruments and equipment. Existing online mass spectrometry systems (https: / / doi.org / 10.1021 / acs.analchem.6b03975; https: / / doi.org / 10.1016 / j.jchromb.2015.03.022; https: / / doi.org / 10.1016 / j.ab.2004.08.010) use a solution of fixed reaction volume and changing flow rate to control reaction time. This solution relies on internal standards, has dead volume interference, a large range of signal intensity fluctuations, and does not consider the need for photoreaction coupling. The current monitoring system has limited continuity and low data throughput, and it is difficult to meet the requirements of high data demand and multiple conditional dimensions in kinetic research. To this end, the utility model comprehensively optimizes online photochemical reaction monitoring. Utility Model Content

[0003] The purpose of the utility model is to provide an online photoreaction monitoring system that can systematically adjust light intensity and irradiation time to conduct mechanism research on small molecule optical probes, in order to solve the problems that the current commercially available photocrosslinking instruments cannot be automated, have fixed and inconsistent parameters, and have no online monitoring equipment.

[0004] In order to achieve the above technical purpose, the utility model adopts the following technical solutions:

[0005] A multi-dimensional online photoreaction monitoring system is characterized in that it includes a sample pump, a transparent pipeline, a programmable LED array and a mass spectrometer, wherein the front end of the transparent pipeline is connected to the sample pump, the rear end is connected to the mass spectrometer, the middle is a serpentine bending area, and the uniform light field generated by the programmable LED array illuminates the serpentine bending area; the reaction sample is pumped into the transparent pipeline by the sample pump, is irradiated by the light field in the serpentine bending area to undergo a photoreaction, and then is input into the mass spectrometer for real-time online monitoring.

[0006] Furthermore, the transparent pipe needs to be resistant to organic solvents and be bendable, and can be a PFA pipe or a PTFE pipe. The inner diameter of the transparent pipe is preferably 0.2 to 1 mm, and the total length of the serpentine bending area is 0.5 to 1.5 meters.

[0007] Furthermore, the programmable LED array is connected to an adjustable constant current power supply controlled by a host computer. The programmable LED array illuminates the serpentine bending area of ​​the transparent pipe, generates a pulse light field through a pulse width modulation (PWM) signal to adjust the illumination time t, and generates brightness changes through the adjustable constant current power supply to adjust the light power density [hv].

[0008] The sample pump is preferably a constant flow pump that can provide a pressure of more than 1 MPa, such as Shimadzu Nexera XR LC-30AD.

[0009] Furthermore, to avoid light reaction before the uniform light field generated by the programmable LED array, the sample pump is connected to the transparent pipeline via an opaque pipeline, which can be a PEEK tube, a PTFE tube or a stainless steel tube.

[0010] The multi-dimensional photoreaction online monitoring system of the utility model can be used to analyze the photoreaction mechanism, and the implementation method is as follows:

[0011] 1. Online monitoring of the mass spectrometry signal intensity of reactants, intermediates and products under different light power density [hv], illumination time t or different wavelength LED illumination;

[0012] 2. Perform mathematical fitting on the multi-dimensional information to verify the photoreaction mechanism and obtain the kinetic parameters.

[0013] As a preferred solution, the sample is pumped into the PFA line at a constant flow rate to improve the stability of subsequent signals and reduce noise.

[0014] As a preferred solution, continuously adjustable illumination time t and light power density [hv] are achieved through a programmable LED array.

[0015] As a preferred solution, real-time multiple reaction monitoring (MRM) is performed on the samples input into the mass spectrometer. MRM-MS improves its sensitivity and reduces noise.

[0016] As a preferred solution, the possible reaction mechanism is pre-investigated before the experiment, the kinetic characteristic curve is calculated, and the kinetic curve and parameters obtained from the experiment are compared to determine the rationality of the kinetic mechanism.

[0017] In an embodiment of the present invention, the multi-dimensional photoreaction online monitoring system of the utility model is used to quantitatively evaluate the photolysis and photoreaction mechanism of diaziridine. In addition to analyzing the photolysis of alkyl diaziridines, the system can also analyze the kinetics of other photoreactions online.

[0018] The utility model provides a multi-dimensional photoreaction online monitoring system, in which the photoreaction is carried out in a transparent pipeline arranged in a serpentine bend under a uniform light field, and the sample is injected at a constant flow rate; both the irradiation time t and the light power density [hv] can be modulated to generate a large number of data points, thereby allowing the fitting of kinetic parameters (insets); the reaction mixture is then injected into a mass spectrometer for real-time multiple reaction monitoring (MRM) analysis; the light field generated by the programmable LED array is uniform and stable, and is simultaneously regulated by pulse width modulation (PWM) to regulate the irradiation time t and the light power density [hv] to be modulated by an adjustable constant current power supply, thereby solving the problems of variable flow rate online mass spectrometry monitoring requiring an internal standard, unstable signals, a small adjustable range, and a large dead time, accelerating the data acquisition speed, and providing an effective solution for the study of the photochemical kinetic mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a structural schematic diagram of an online optical reaction monitoring system of the utility model, comprising: 1-a sample container, 2-a sample pump, 3-a PFA tube, 4-a programmable LED array, and 5-a mass spectrometer.

[0020] Figure 2 It is a schematic diagram of the uniformity of the light field under the illumination of a programmable LED array device, and the serpentine-shaped PFA tube is in the center of the uniform light field.

[0021] Figure 3 The following are 31 patterns of the pulse width modulation (PWM) signal modulating the illumination time t. By setting the duty cycle uniformly, the illumination time can be linearly adjusted.

[0022] Figure 4 It is a diagram of 256 current modes of adjustable constant current power supply modulating optical power density [hv]. The adjustable range of optical power density [hv] is 0.1~289mW / cm 2 .

[0023] Figure 5 This is a possible photolysis mechanism model of alkyldiaziridine and its computational simulation diagram.

[0024] Figure 6 This is a possible photolysis mechanism analysis diagram of alkyl diaziridine, where a fits 242mW / cm 2 a) The photolysis of A and the generation of D under the condition of photolysis of A and D under the condition of photolysis of D; b) (k1+k3) and k2 are obtained by linear fitting of all experimental data; c) The k1 / (k1+k3) obtained by fitting is between 0.85 and 0.92. All experiments were repeated three times, and the standard deviation is given. The solid line is the fitting curve. DETAILED DESCRIPTION

[0025] The following describes the composition, structure and application of the present invention through embodiments in conjunction with the accompanying drawings, but does not limit the protection scope of the present invention in any way.

[0026] like Figure 1 As shown, the liquid sample in the sample container 1 is injected into the PFA tube 3 at a constant flow rate under the action of the sample pump 2, and reacts continuously in the PFA tube under the stimulation of ultraviolet light. The programmable LED array 4 provides a uniform light field, and the PFA tube bends in a serpentine shape under the uniform light field. The programmable LED array 4 bears a vital responsibility in the two-dimensional illumination adjustment: it generates a pulse light field through a pulse width modulation (PWM) signal to adjust the illumination time t, and generates brightness changes through an adjustable constant current power supply to adjust the light power density [hv]. After the sample undergoes a rapid light reaction in the PFA tube 3, it is injected into a mass spectrometer (MS) for real-time multiple reaction monitoring (MRM). MRM-MS improves its sensitivity and reduces noise.

[0027] In this example, a Shimadzu Nexera XR LC-30AD pump was used to pump the sample at a rate of 0.200 mL / min. The solution was injected into a transparent PFA tube (1 / 16 inch outer diameter, 0.5 mm inner diameter; total length of the serpentine bending area was 1 meter, volume 0.196 mL) through an opaque PEEK tube for photoreaction. A programmable LED array and an adjustable constant current power supply were customized from Lightwells (Shenzhen, China). The adjustable constant current power supply and pulse width modulation (PWM) signal were controlled by a Raspberry Pi 4B (https: / / www.raspberrypi.com). After the photoreaction occurred in the PFA tube, the Figure 3 and Figure 4 The design shown in the figure linearly adjusts the illumination time, and the adjustable range of light power density [hv] is 0.1~289mW / cm 2 The solution was continuously injected into Shimadzu 8050MS for real-time multiple reaction monitoring (MRM) analysis (Table 1 gives the experimental parameters). Data analysis was performed using Lab Solutions (version 5.91) from Shimadzu Corporation.

[0028] Table 1 Parameters used for online MS MRM monitoring of photolysis of alkyl diaziridines

[0029]

[0030]

[0031] Figure 5Four possible conversion pathways and kinetic mechanisms of previously reported alkyl diaziridines are shown, in which diazo intermediate B and carbene intermediate C originate from the photolysis of alkyl diaziridine A and finally give the main quenching product D. The corresponding kinetic curves can be obtained by calculation.

[0032] [A] t =[A]0exp(-(k1+k3)[hv]t)

[0033]

[0034]

[0035] Wherein, hv represents the light power density, t represents the illumination time, k1 represents the photolysis kinetic constant of alkyl diaziridine A, k2 represents the kinetic constant of the conversion of diazo intermediate to carbene intermediate C, k3 represents the kinetic constant of direct photolysis of alkyl diaziridine A to produce C, k ox The kinetic constant for the oxidation of the carbene intermediate C by air to the oxidation product D, k others It represents the kinetic constant of the quenching of carbene intermediate C to produce products other than oxidation product D. Model I has k3=0; except for model III with k2'=k2 / [hv], the other models have k2'=k2; model IV has k2=0.

[0036] Sulfo-SDA (sulfo-NHS-diazirine, an alkyl diaziridine molecule) was dissolved in 100 mM dimethyl sulfoxide and diluted with acetonitrile to a final concentration of 0.25 μM. The flow rate through the PFA tube was set to 0.200 mL / min, which took 58.9 seconds (~1 minute). The illumination time t was linearly controlled by PWM at a constant light power density [hv]. Reactant A and product D ( Figure 5 ) were monitored in MRM mode and the ion intensities were normalized before fitting. Linear regression was used to fit the changes of A and D over time to obtain (k1+k3)[hv] and k2[hv]; the experiment was repeated at different multiple light power densities [hv].

[0037] We used MRM-MS to monitor the photoreaction in real time and found that the increase in the MS signal of D over time could not be fitted into a single exponential function ( Figure 6 ), which helps us to rule out Model IV. On the other hand, increasing the optical power density [hv] from 101mW / cm 2 Increased to 242mW / cm 2,D curves hardly changed, which further helped to exclude model III. The fitting results showed that k1 / (k1+k3) was between 0.85 and 0.92, so model II was the best kinetic mechanism model to describe the photolysis mechanism of alkyl diaziridine.

Claims

1. A multi-dimensional online optical reaction monitoring system, characterized in that: The invention comprises a sample pump, a transparent pipeline, a programmable LED array and a mass spectrometer, wherein the front end of the transparent pipeline is connected to the sample pump, the rear end is connected to the mass spectrometer, the middle is a serpentine bending area, and the uniform light field generated by the programmable LED array illuminates the serpentine bending area; the reaction sample is pumped into the transparent pipeline by the sample pump, is irradiated by the light field in the serpentine bending area for light reaction, and then is input into the mass spectrometer for real-time online monitoring.

2. The multi-dimensional online optical reaction monitoring system according to claim 1, characterized in that: The transparent tube is a PFA tube or a PTFE tube.

3. The multi-dimensional online optical reaction monitoring system according to claim 1, characterized in that: The inner diameter of the transparent pipe is 0.2 to 1 mm, and the total length of the serpentine bending area is 0.5 to 1.5 meters.

4. The multi-dimensional online optical reaction monitoring system according to claim 1, characterized in that: The multi-dimensional online light reaction monitoring system also includes an adjustable constant current power supply, and the programmable LED array is connected to the adjustable constant current power supply.

5. The multi-dimensional online optical reaction monitoring system according to claim 1, characterized in that: The sample pump is a constant flow pump.

6. The multi-dimensional online optical reaction monitoring system according to claim 1, characterized in that: The sample pump is connected to the transparent pipeline through a section of opaque pipeline.

7. The multi-dimensional online optical reaction monitoring system according to claim 6, characterized in that: The opaque pipe is a PEEK pipe, a PTFE pipe or a stainless steel pipe.

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