3D metal printing ultraviolet detector and application thereof in real-time monitoring of column chromatography fraction collection

Abstract

The invention discloses a 3D metal printing ultraviolet detector and application of the 3D metal printing ultraviolet detector in real-time monitoring of column chromatography fraction collection. The 3D metal printing ultraviolet detector can be directly connected with a chromatographic column and can be used for monitoring fractions in a column chromatography process in real time; especially, when the separated mixed substance is colorless but has an ultraviolet absorption characteristic, whether the substance is eluted or not cannot be judged by naked eyes, and can be monitored by the ultraviolet detector. According to the method, the problem that the tedious steps of continuously receiving fractions and then carrying out thin-layer chromatography identification in the existing column chromatography are solved, whether substances are separated or not can be detected through the 3D metal printing ultraviolet detector, and then fraction collection is carried out, so that time is saved. In addition, the 3D metal printing ultraviolet detector has the advantages of being simple in light path design, low in cost, easy to assemble, small in size and capable of tolerating low-polarity organic solvents.

Description

Technical Field

[0001] The present invention belongs to the technical field of column chromatography detection. Specifically, it relates to a 3D metal printed ultraviolet detector and its application in real-time monitoring of column chromatography fraction collection. Background Art

[0002] Chromatography columns are commonly used for separating and purifying mixtures. However, in experiments using chromatography columns, when qualitatively analyzing the separated substances, thin layer chromatography is usually used, which requires continuously receiving fractions and then identifying them one by one, and the process is rather cumbersome and time-consuming. Patents with publication numbers CN202185180U and CN201658863U have developed an ultraviolet-transmitting chromatography column device for separating and purifying mixed chemical components. By filling a fluorescent medium material in the chromatography column and irradiating it with an ultraviolet lamp, the separated substances show different fluorescent color bands, enabling real-time observation of the separation situation inside the column. This helps to solve the problems that the separation process of the current chromatography column cannot be observed with the naked eye in real time and cannot be adjusted online quickly and conveniently. However, this requires the use of a special glass chromatography column that can transmit ultraviolet light and also requires the use of a material with a fluorescent medium as the filler, and the overall device is relatively large and costly.

[0003] Although the currently existing ultraviolet detectors have very good detection performance, they are relatively large in size, most of the parts used for assembling the detectors are relatively expensive, and the optical path design is also a bit complex. Currently, there is no 3D metal printed ultraviolet detector that can be directly connected to a chromatography column to monitor the elution process of the chromatography column and can determine the end point of substance elution. Therefore, it is of great significance to provide a 3D metal printed ultraviolet detector with a small volume, simple preparation, and low cost, which is helpful for real-time monitoring of the substance elution process of a conventional chromatography column. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art and provide a 3D metal printed ultraviolet detector.

[0005] The second purpose of the present invention is to provide the application of the 3D metal printed detector in real-time monitoring of column chromatography fraction collection.

[0006] The above purposes of the present invention are achieved by the following technical solutions:

[0007] The present invention first provides a 3D metal printed ultraviolet detector, which includes an ultraviolet light source, a flow cell, a photoelectric signal conversion component, a signal processing component, a chromatography column interface, and a fraction collection port; a liquid inlet is provided at the top of the flow cell, a liquid outlet is provided at the bottom of the flow cell, and light passing ports are provided on both sides of the flow cell and quartz glass sheets are embedded therein; the chromatography column interface is connected to the liquid inlet of the flow cell, and the liquid outlet of the flow cell is connected to the fraction collection port; the ultraviolet light source and the photoelectric signal conversion component are respectively arranged on both sides of the flow cell, the ultraviolet light source is used to emit ultraviolet light, and the ultraviolet light passes through the quartz glass sheets on both sides of the flow cell and is received by the photoelectric signal conversion component, and after being received, the photoelectric signal conversion component converts the optical signal into an electrical signal for output; the signal processing component is connected to the photoelectric signal conversion component and is used to receive and record the output electrical signal data.

[0008] The 3D metal printed ultraviolet detector provided by the present invention can be directly connected to a chromatography column and can monitor the fractions in the column chromatography process in real time; in addition, compared with the existing ultraviolet detectors, the optical path design of the 3D metal printed ultraviolet detector of the present invention is simpler. The ultraviolet detector provided by the present invention has the characteristics of low cost, simple assembly, and small volume, and only ordinary chromatography columns and ordinary chromatographic packings are needed in the use process.

[0009] When in use, the 3D metal printed ultraviolet detector is connected to a chromatography column to monitor the fractions in the column chromatography process in real time. When a substance with light absorption characteristics is eluted, the reading output by the signal processing component decreases, and an inverted peak will appear in the recorded electrical signal change curve; when the reading value returns to the baseline position, it indicates that the substance has been completely eluted. Therefore, the fraction collection can be started only when the reading value decreases, so as to realize the collection of substances, saving the time required for continuous fraction collection and thin layer chromatography identification. Experiments have proved that the 3D metal printed ultraviolet detector has good stability and linearity (R 2 > 0.99). Two systems, namely, the methyl orange - methylene blue mixed solution whose separation effect can be observed with the naked eye and the salicylic acid - aspirin mixed solution whose separation effect cannot be observed with the naked eye, are selected to verify the feasibility of the 3D metal printed ultraviolet detector. The results prove that the 3D metal printed ultraviolet detector can monitor the fractions of the mixture separation in real time, verifying its feasibility. When a substance with light absorption characteristics is eluted, the reading of the signal processing component connected to the detector decreases, and the fraction collection can be realized, so as to realize the positioning collection of substances.

[0010] Furthermore, the flow cell, the chromatography column interface, and the fraction collection port are of an integrated structure.

[0011] Further, the flow cell, chromatography column interface, and fraction collection port are made of an aluminum alloy outer casing printed by a 3D metal printer. Using 3D printing technology, the structure is simple and the cost is low. The UV detector printed by 3D metal is small in size and the cost of the parts used is low.

[0012] Further, the chromatography column interface is a large hollow cylinder; the fraction collection port is a small hollow cylinder. Thus, the solution flowing out of the chromatography column can not only fill the flow cell but also flow out from the fraction collection port at the lower end of the flow cell, so as to achieve the purpose of detecting the solution and replacing the solution in the flow cell.

[0013] Further, the wavelength range of the ultraviolet light emitted by the ultraviolet light source is 250 - 260 nm.

[0014] Further, the cell body of the flow cell is a cuboid.

[0015] Further, the volume of the flow cell is 12 - 48 mm 3 。

[0016] Preferably, the volume of the flow cell is 12 mm 3 。

[0017] Preferably, the optical path of the flow cell is 3 mm.

[0018] Further, the ultraviolet light source is connected to the flow cell through a fixture, and the fixture is used to fix the distance between the ultraviolet light source and the flow cell.

[0019] Preferably, the distance between the ultraviolet light source and the flow cell is 4 - 5 mm.

[0020] Further, the optoelectronic signal conversion component is a silicon photocell.

[0021] Further, the signal processing component is a voltmeter. The voltmeter can also be connected to a computer to record the voltage change curve through software.

[0022] Further, a heat dissipation component is fixed on the ultraviolet light source.

[0023] Preferably, the heat dissipation component is selected from any one or more of a Peltier cooler, a heat sink, or a cooling fan.

[0024] Preferably, the heat dissipation component is composed of a Peltier cooler, a heat sink, and a cooling fan; the ultraviolet light source is fixed on the Peltier cooler, and the other side of the Peltier cooler is sequentially connected to the heat sink and the cooling fan.

[0025] The present invention provides the application of the above-mentioned 3D metal printing UV detector in the real-time monitoring of column chromatography fraction collection.

[0026] Further, the application is to monitor in real time the elution of a colorless substance with ultraviolet absorption characteristics during the column chromatography process.

[0027] The present invention also provides a method for collecting column chromatography fractions based on the 3D metal printed ultraviolet detector described in any one of the above, including connecting the chromatography column to the chromatography column interface of the 3D metal printed ultraviolet detector, adding the sample to be separated to the chromatography column to start elution, starting to collect fractions when the electrical signal data output by the signal processing component becomes smaller, and stopping collection when the electrical signal data output by the signal processing component returns to the baseline. That is, it only needs to start collecting when a substance is detected to be eluted by ultraviolet detection and stop collecting when the substance is detected to have been completely eluted, greatly reducing the workload.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides a 3D metal printed ultraviolet detector that can be directly connected to the chromatography column, which helps to monitor the fractions in the column chromatography process in real time; especially when the separated mixture is colorless but has ultraviolet absorption characteristics and it is impossible to visually judge whether the substance is eluted, it can be monitored by the 3D metal printed ultraviolet detector. The present invention solves the problem of the cumbersome steps of continuously receiving fractions and then performing thin layer chromatography identification in current column chromatography, and can detect whether a substance is separated by the 3D metal printed ultraviolet detector before collecting the fractions, saving time; at the same time, it solves the problem that the prior art needs to use glass made of special materials that absorb ultraviolet rays and materials with fluorescent media as fillers, and only ordinary chromatography columns and fillers are required. In addition, compared with the existing ultraviolet detectors, the 3D metal printed ultraviolet detector has a simpler optical path design, and has the characteristics of low cost, simple assembly, small volume and resistance to low-polarity organic solvents. Description of the Drawings

[0030] Figure 1 It is a schematic structural diagram of a 3D metal printed ultraviolet detector; wherein, 1 is an ultraviolet light source, 2 is a flow cell, 21 is a liquid inlet, 22 is a liquid outlet, 23 is quartz glass, 3 is a chromatography column interface, 4 is a fraction collection port, 5 is a photoelectric signal conversion device, and 6 is a signal processing component.

[0031] Figure 2 It is a component picture and usage diagram of a 3D metal printed ultraviolet detector; wherein, Figure 2In Figure A is a schematic diagram of the overall device of the 3D metal-printed ultraviolet detector, where a is a cooling fan, b is an aluminum heat sink, c is a semiconductor refrigeration chip, d is an ultraviolet lamp bead with a wavelength of 250 - 260 nm, e is a fixture for the ultraviolet lamp, f and h are quartz glass sheets, g is the main body of the 3D metal-printed ultraviolet detector, and i is a silicon photocell; Figure B is a physical diagram (a) when the chromatography column is connected to the 3D metal-printed ultraviolet detector during operation and a physical diagram (b) of the main body part of the 3D-printed metal ultraviolet detector; Figure C is a schematic diagram of the cross-sectional details of the main body device of the 3D metal-printed ultraviolet detector.

[0032] Figure 3 are the performance test results of the 3D metal-printed ultraviolet detector; among them, Figure 3 in Figure A shows the voltage change of the silicon photocell and the ultraviolet lamp during continuous operation for 60 min, and Figure B shows schematic diagrams of the 3D metal-printed ultraviolet detector with a flow cell volume of 12 mm 3 (a), 24 mm 3 (b), 48 mm 3 (c), and Figure C shows the linear range of the 3D metal-printed ultraviolet detector with a flow cell volume of 12 mm 3 (a), 24 mm 3 (b), 48 mm 3 (c).

[0033] Figure 4 are the test results of the solution replacement time of the 3D metal-printed ultraviolet detector with flow cell volumes of 12 mm 3 (A), 24 mm 3 (B), 48 mm 3 (C).

[0034] Figure 5 is the process of using the 3D metal-printed ultraviolet detector to monitor the separation process of a methyl orange and methylene blue mixed solution on a silica gel chromatography column; among them, Figure 5 in Figure A shows the process diagrams of the methyl orange and methylene blue mixed solution just being loaded onto the silica gel chromatography column (a), eluting methyl orange (b), and eluting methylene blue (c); Figure B shows the real-time monitoring curve of the 3D metal-printed ultraviolet detector for the fractions during the column chromatography separation process of the methyl orange and methylene blue mixed solution.

[0035] Figure 6 is a schematic flow diagram of the real-time monitoring of the separation of salicylic acid and aspirin on a silica gel chromatography column using a 3D-printed ultraviolet detector.

[0036] Figure 7 is the process of using the 3D metal-printed ultraviolet detector to monitor the separation process of salicylic acid and aspirin on a silica gel chromatography column; among them, Figure 7In Figure A, it is the real-time monitoring curve of the separation of salicylic acid and aspirin on a silica gel chromatography column by a 3D metal-printed ultraviolet detector; in Figure B, it is the identification of the components of the collected fractions by thin-layer chromatography; in Figure C, it is the determination of the fractions that show ultraviolet absorption in the detection of the 3D metal-printed ultraviolet detector but cannot be identified by thin-layer chromatography by high-performance liquid chromatography; in Figure D, it is the high-performance liquid chromatography chart of the salicylic acid standard product. Detailed implementation mode

[0037] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0039] Example 1

[0040] The composition of the 3D metal-printed ultraviolet detector is as Figure 1 shown, including an ultraviolet light source 1, a flow cell 2, a chromatography column interface 3, a fraction collection port 4, a photoelectric signal conversion component 5, and a signal processing component 6; a liquid inlet 21 is provided at the top of the flow cell 2, a liquid outlet 22 is provided at the bottom, and light-passing ports are provided on both the left and right sides and quartz glass sheets 23 are embedded; the chromatography column interface 3 is connected to the liquid inlet 21 of the flow cell, and the liquid outlet 22 of the flow cell is connected to the fraction collection port 4; the ultraviolet light source 1 is used to emit ultraviolet light, and the ultraviolet light passes through the quartz glass sheets 23 on both sides of the flow cell 2 and is received by the photoelectric signal conversion component 5, and after the photoelectric signal conversion component 5 receives it, the optical signal is converted and output as an electrical signal; the signal processing component 6 is connected to the photoelectric signal conversion component 5 and is used to receive and record the output electrical signal data. Among them, the ultraviolet light source 1 is an ultraviolet lamp bead of 250-260 nm, the photoelectric signal conversion component 5 is a silicon photocell, and the signal processing component 6 is a voltmeter and the computer connected thereto.

[0041] The 3D metal-printed ultraviolet detector is prepared by 3D metal printing technology, and the specific preparation process is as follows:

[0042] The main body part of the 3D metal-printed ultraviolet detector is an aluminum alloy shell printed by a 3D metal printer, which is mainly composed of a large hollow cylinder, a small hollow cylinder, and a cuboid designed with light-passing ports and a flow cell. The internal cross-sectional detail drawing of the cuboid part is as Figure 2 shown in Figures C(a) and (b). The flow cell 2 is composed of a space of 2 mm×3 mm×2 mm, and two quartz glass sheets 23 with a diameter of 7 mm and a thickness of 1 mm are embedded on the left and right ( Figure 2In A(f) and (h), such a design can not only allow the light of the ultraviolet lamp to pass through but also form a flow cell; the small hollow cylinder at the lower end of the detector is the liquid outlet 22. Since the upper end of the detector, i.e., the chromatography column interface 3, just connects to the lower end of the chromatography column, it has a certain sealing property. The hollow cylinder at the lower end, i.e., the fraction collection port 4, has a smaller diameter. Therefore, after opening the piston of the chromatography column, the solution can not only fill the flow cell 2 but also flow out from the fraction collection port 4 at the lower end of the detector, thus achieving the purpose of detecting the solution and replacing the solution in the flow cell. The internal cross-sectional detail diagram of the cuboid part is as shown in Figure 2 C(a) and (b) in. The ultraviolet light source 1 used in the 3D metal printed ultraviolet detector is an ultraviolet lamp bead with a wavelength in the range of 250 - 260 nm ( Figure 2 in A(d)). Since the ultraviolet lamp works for a long time, it will generate heat, which will affect the light-emitting performance and stability of the ultraviolet lamp. Therefore, the ultraviolet lamp is adhered to the semiconductor refrigeration chip ( Figure 2 in A(c)), and an aluminum heat sink is adhered to the other side of the semiconductor refrigeration chip ( Figure 2 in A(b)), and then a cooling fan is added ( Figure 2 in A(a)), which can well achieve the effect of dissipating heat from the ultraviolet lamp. In order to fix the distance between the ultraviolet lamp and the flow cell, a fixture is printed by a 3D printer using PLA material ( Figure 2 in A(e)). One end is adhered to one side of the detector, and the other end is installed on the semiconductor refrigeration chip, thus fixing the distance between the ultraviolet lamp and the flow cell, and the distance is approximately 4.4 mm. A silicon photocell is placed on the other side of the flow cell ( Figure 2 in A(i)) and connected to an ammeter. The silicon photocell can convert the optical signal into an electrical signal and display it on the ammeter in real time. Connecting to a computer, the data can be recorded in real time through software analysis. The change in the voltage value indicates that the light intensity received by the photosensitive surface on the silicon photocell changes. When the solution flowing through the flow cell contains substances that absorb ultraviolet light, the substances absorb the ultraviolet light, so the ultraviolet light intensity irradiating on the photosensitive surface of the silicon photocell through the flow cell weakens, and the ammeter shows a voltage drop. Therefore, when the value of the voltmeter decreases, it indicates that a substance with ultraviolet absorption is flowing into the flow cell. The overall device of the 3D metal printed ultraviolet detector is as shown in Figure 2 B(a) in. During the experiment, the entire device is clamped with a clip and installed on an iron stand for easy connection to the chromatography column, as shown in Figure 2 B(b) in. The lower end of the chromatography column can just be embedded into the large hollow cylinder at the upper end of the designed detector, that is, the lower end of the chromatography column can be fitted with the chromatography column interface 3 at the upper end of the flow cell 2.

[0043] Example 2 Performance determination of the 3D metal printed ultraviolet detector

[0044] 1. Test on the stability of the silicon photocell and the ultraviolet lamp:

[0045] According to Example 1, first assemble the entire device of the 3D metal printed ultraviolet detector. Install the semiconductor refrigeration sheet with the ultraviolet lamp on the detector with the holder, and through the other end of the detector, observe whether the ultraviolet lamp beads are in the center through the flow cell. Then install the silicon phototube on the device and connect the voltmeter. In order to detect the short-term stability of the silicon photocell within 60 minutes, after installing the entire device, without turning on the ultraviolet lamp, observe the change of the voltage value within 60 minutes and record the voltage value every 5 minutes. When the ultraviolet lamp is used for a long time in the experiment, if its heat dissipation conditions cannot be met, the temperature of the ultraviolet lamp will increase, resulting in a decrease in the ultraviolet light intensity. Test whether the externally added semiconductor refrigeration sheet, aluminum heat sink, and cooling fan meet its heat dissipation requirements. Therefore, it is necessary to detect the stability of the ultraviolet lamp. Turn on the 9V power supply connected to the ultraviolet lamp, adjust the voltage of the semiconductor refrigeration sheet and the cooling fan to about 12V, observe the change of the voltage within 60 minutes, and record the voltage value every 5 minutes.

[0046] The results of the stability of the silicon photocell and the ultraviolet lamp are as Figure 3 shown in A. From the results, it can be seen that the silicon photocell has good stability, the amplitude of the voltage value fluctuation is not large, the average voltage value is 0.00083V, and the standard deviation is 0.00058. The ultraviolet lamp has been continuously working for 60 minutes, and its luminous intensity has not changed significantly. The voltage value displayed on the voltmeter when the silicon photocell converts the optical signal into an electrical signal fluctuates around 0.272V, its average voltage value is 0.2718V, and the standard deviation is 0.00072. This shows that the ultraviolet lamp still has good luminous stability after continuous operation for 60 minutes, and at the same time, it also shows that the equipped semiconductor refrigeration sheet and cooling fan have a good heat dissipation effect, and the voltage of the connected power supply is also appropriate and will not be overloaded.

[0047] 2. Determine the linear range and detection limit for detectors with different flow cell volumes:

[0048] Designed flow cells with a consistent optical path of 3mm and volumes of 12mm 3 , 24mm 3 , 48mm 3 detectors ( Figure 3 shown in B).

[0049] Preparation of acetaminophen solutions with different concentrations: Weigh 0.0502 g of acetaminophen and dissolve it in 50 mL of 95% ethanol solution to obtain an acetaminophen ethanol solution with a concentration of 1004 μg / mL as the stock solution. Dilute the acetaminophen ethanol solution with a concentration of 1004 μg / mL to obtain a series of acetaminophen ethanol solutions with concentrations of 40.16 μg / mL, 30.12 μg / mL, 25.1 μg / mL, 20.08 μg / mL, 15.06 μg / mL, and 10.04 μg / mL respectively.

[0050] After assembling the device, without connecting it to the chromatography column, directly test the linear range of the detector device. First, fill the flow cell of the detector device with 95% ethanol to adjust the baseline and record the voltage value at this time. Then, add acetaminophen ethanol solutions with different concentrations to the flow cell of the detector. After rinsing it three times with the solution, record the voltage value again. Note that it is necessary to adjust the baseline with 95% ethanol before measuring each solution with a different concentration. Finally, subtract the measured voltage value of the acetaminophen ethanol solution from the corresponding baseline voltage value to obtain the concentration-voltage difference of each acetaminophen ethanol solution. Plot the relationship between concentration and voltage difference, and through linear fitting, the linear graph of this detector can be obtained.

[0051] In addition to the performance investigation of the linear range, another investigation index for the performance of the 3D metal printing ultraviolet detector is the detection limit, and the calculation formula for the detection limit is as follows:

[0052] Limit of detection LOD:

[0053]

[0054]

[0055] In the formula:

[0056] σ: Standard deviation;

[0057] S: Slope of the standard linear curve;

[0058] I 0i : Measured value of a single determination of the blank solution;

[0059] Average value of the measurement;

[0060] n: Number of measurements.

[0061] The results of exploring the linear range are as shown in Figure 3 shown in C, for a flow cell volume of 12 mm 3The detector was used for measurement. Linear fitting was performed on the ethanol solutions of paracetamol at different concentrations and the voltage values measured by the detector, and the linear equation obtained was y = 0.0009x + 0.007, R 2 was 0.9961. The 3D metal printed UV detector had a good linear relationship in the determination of paracetamol concentration in the range of 10.04 μg / mL to 40.16 μg / mL. The voltage values of 95% ethanol were measured in parallel three times, and the values were 0.294 V, 0.295 V, and 0.295 V. The calculated standard deviation was 0.00058, and the calculated LOD = 2.13 μg / mL.

[0062] Using the same method, a linearity test was carried out on a 3D metal printed UV detector with a flow cell volume of 24 mm 3 . The ethanol solution of paracetamol had a good linear relationship at a concentration range of 10.04 μg / mL to 30.12 μg / mL, y = 0.0011x + 0.0071, R 2 was 0.9919. The voltage values of 95% ethanol were measured in parallel three times, and the values were 0.292 V, 0.293 V, and 0.293 V. The calculated standard deviation was 0.00058, and the LOD = 1.74 μg / mL.

[0063] For the 3D metal printed UV detector device with a flow cell volume of 48 mm 3 , the ethanol solution of paracetamol had a good linear relationship at a concentration range of 10.04 μg / mL to 40.16 μg / mL, y = 0.0008x + 0.0069, R 2 was 0.9967. The voltage values of 95% ethanol were measured in parallel three times, and the values were 0.302 V, 0.302 V, and 0.303 V. The calculated standard deviation was 0.00058, and the LOD = 2.39 μg / mL.

[0064] Therefore, considering the linearity of the ethanol solutions of paracetamol measured by the three devices with different flow cell volumes, all showed good linear relationships (R 2 > 0.99).

[0065] Example 3: Test on the solution replacement time of 3D metal printed UV detectors with different volumes of flow cells

[0066] When the concentration of the detected solution is low, after entering the flow cell of the detector, the concentration will be diluted to a certain extent. If the volume of the flow cell is smaller, the dilution multiple can be greatly reduced, and when the volume is small, the replacement speed of the solution in the flow cell can be accelerated, thereby improving the response recovery speed of the detector. Therefore, further, the solution replacement times of 3D metal printed UV detectors with different flow cell volumes are compared. When the solution enters the flow cell, once the substance with UV absorption characteristics passes through the flow cell, the silicon photocell can detect the weakening of the intensity of the UV light, which is manifested as a decrease in the voltage value. As the solution flows, the concentration of the solution with the light-absorbing substance in the flow cell gradually decreases, and at this time, the voltage of the electrical signal conducted by the silicon photocell gradually increases until the solution in the flow cell is completely replaced by the solution without UV absorption characteristics, and at this time, the voltage value returns to the baseline. The size of the flow cell volume is related to the solution replacement speed. The smaller the flow cell volume, the faster the solution replacement speed, and the shorter the detector response recovery time. Therefore, for the flow cell volume of 12mm 3 , 24mm 3 , 48mm 3 of the 3D metal printed UV detector are tested by comparing the length of time it takes for the voltage value to return to the voltage value at the baseline after the substance with UV absorption passes through. The specific method is as follows:

[0067] Weigh 0.0509 g of methylene blue and dissolve it in 50 mL of 95% ethanol solution to obtain methylene blue solution. Then connect the 3D metal printed UV detector to the lower end of the chromatography column, add 95% ethanol to the chromatography column, and adjust the solution to drip at a speed of 2 drops per second. Use the voltage value measured when 95% ethanol flows into the flow cell as the baseline. After waiting for the baseline to be stable, start the measurement. When the liquid level of the 95% ethanol solution drops to the position of the chromatography column sand core, use a 5 mL pipette to suck 2 mL of methylene blue solution and add it to the chromatography column. Note that during the addition process, do not touch the wall, and try to insert the pipette into the chromatography column vertically for addition. When the liquid level of the methylene blue solution drops to the position of the sand core, add 1 mL of 95% ethanol solution to start elution. Then wait for the liquid level to drop to the position of the sand core again, and add 1 mL of 95% ethanol solution. Repeat the operation multiple times until the voltage value returns to near the baseline and remains unchanged for 30 s, which means the replacement of the solution in the flow cell is completed. Measure three times in parallel, calculate the time for the liquid replacement in the flow cell, and take the average value. Similarly, measure 3D metal printed UV detectors with different other flow cell volumes.

[0068] The results are as Figure 4 shown. When the substance with UV absorption passes through the flow cell, the voltage readings of the detectors with different flow cell volumes all immediately decrease. After the substance with UV absorption passes through, the voltage readings start to increase. It can be intuitively seen from the trend of the curves in the figure that the recovery curve of the flow cell with a volume of 48mm 3 is relatively flat (Figure 4 In C), the flow cell volume is 12 mm 3 The recovery curve is steeper ( Figure 4 In A). It can be seen from the calculated results that the flow cell volume is 12 mm 3 For the 3D metal printed UV detector, when the voltage reading returns to the baseline value, the time is the shortest, only 83 s. The second is the detector with a volume of 24 mm 3 of the 3D metal printed UV detector ( Figure 4 In B), the required time is 108 s. Finally, it is the detector with a volume of 48 mm 3 of the 3D metal printed UV detector, and the time to return to the baseline voltage value is longer, 172 s. Combining the above results analysis, under the condition that the linear performance of the detectors with three different volume flow cells has no significant difference, the 3D metal printed UV detector with a flow cell volume of 12 mm 3 exhibits excellent response and recovery performance. The smaller the flow cell volume, the faster the replacement speed of the solution, and the faster the detector's response reaches the baseline. Therefore, for subsequent experiments, the 3D metal printed UV detector with a flow cell volume of 12 mm 3 is used for the test to achieve the best effect.

[0069] Example 4 Real-time monitoring of the column chromatography separation fractions of methyl orange - methylene blue mixed solution by 3D metal printed UV detector

[0070] Column chromatography separation of methyl orange and methylene blue mixed solution:

[0071] Preparation of eluent: Solution A: Mix ultrapure water and 95% ethanol in a ratio of 1:1. Solution B: Mix 0.2 mol / L hydrochloric acid and 95% ethanol in a ratio of 1:1. Preparation of methyl orange - methylene blue mixed solution: Weigh 0.0092 g of methyl orange and 0.0098 g of methylene blue and dissolve them in 10 mL of Solution A.

[0072] Weigh 4.03 g of silica gel (100 - 140 mesh) into a beaker, add ultrapure water and stir to make the silica gel absorb water and become a paste. Add 1 / 3 column height of ultrapure water to the chromatography column, pour the silica gel in the beaker into the chromatography column while stirring, and at the same time open the piston of the chromatography column, adjust the flow rate to 2 drops per second, gently tap the column body of the chromatography column with an ear bulb to make the plane of the silica gel flat, connect a 3D metal printed ultraviolet detector, and observe whether the flow cell is completely filled. When the liquid level just reaches 1 mm above the silica gel plane, add about 4 mL of solution A, then wait for the liquid level to drop to 1 mm from the silica gel plane, add 70 μL of methyl orange - methylene blue mixed solution. When the mixed solution completely penetrates into the silica gel, immediately add solution A for elution. After the yellow band is completely eluted, add solution B for elution, and at this time, it can be seen that the blue band begins to move down. When the blue band is completely eluted, the separation experiment of the methyl orange - methylene blue mixed solution is completed. Pay attention to observing the change in the reading of the voltmeter during the experiment.

[0073] Figure 5 Figure A shows an experimental diagram of connecting a 3D metal printed ultraviolet detector to the lower end of a chromatography column filled with silica gel and the elution process of adding methyl orange - methylene blue solution. As Figure 5 shown in Figures A(a) and (b), after adding the methyl orange - methylene blue solution, with the elution of solution A, it gradually separates into two colored bands, and the methyl orange in the yellow band is eluted first. Methyl orange is insoluble in organic solvents such as ethanol and soluble in water, and solution A can well desorb methyl orange from silica gel. The blue band is methylene blue ( Figure 5 Figure A(c)), methylene blue is soluble in ethanol and water, and there is a part of its structure equivalent to a quaternary ammonium salt structure, existing in an ionic state. It cannot be desorbed from silica gel only with water or organic solvents with strong polarity. 0.2 mol / L hydrochloric acid is added to solution B, which can enhance the interaction with the quaternary ammonium ions in the methylene blue molecule, thereby desorbing methylene blue from silica gel. Figure 5In Figure B, it is the voltage value change curve detected by the 3D metal printed UV detector connected to the lower end of the chromatography column, which records the voltage value change during the entire process of column chromatography separation of the methyl orange - methylene blue mixed solution. The black part of the curve is the solution without light - absorbing substances, that is, the eluent. When the yellow band is eluted, since the methyl orange in the yellow band can absorb part of the ultraviolet light when passing through the flow - through cell of the 3D metal printed UV detector, at this time, the voltage silicon photocell detects a decrease in the light intensity, manifested as a decrease in the voltage value. As the methyl orange is gradually eluted, the voltage value gradually returns to the baseline value. When changing to elute methylene blue with solution B, at the beginning, methylene blue has not been eluted and flowed into the flow - through cell, and the 3D metal printed UV detector still does not detect the presence of light - absorbing substances, so the voltage value does not change. Subsequently, when the methylene blue in the blue band starts to enter the detector, the value of the voltmeter decreases, proving that methylene blue is eluted. The results show that the 3D metal printed UV detector can well monitor the whereabouts of substances with ultraviolet - absorbing properties on the chromatography column from the beginning of elution to the end of elution. The 3D metal printed UV detector has excellent real - time monitoring performance, can detect in real - time whether there are light - absorbing substances in the solution, and helps to monitor the fractions in the column chromatography process in real - time.

[0074] Example 5 Real - time Monitoring of the Fractions in the Column Chromatography Separation of a Salicylic Acid and Aspirin Mixed Solution by a 3D Metal Printed UV Detector

[0075] In order to further detect the real - time monitoring performance of the 3D metal printed UV detector for column chromatography fractions, the present invention selects the salicylic acid and aspirin system for separation and identification. When separating salicylic acid and aspirin only using a chromatography column, it is impossible to judge in real - time when aspirin is eluted because aspirin is colorless and cannot be judged by the naked eye. Only by continuously collecting many small vials of fractions and then performing thin - layer chromatography (TLC) identification. Such identification is very cumbersome and the workload is very large. However, with the 3D metal printed UV detector, it is possible to monitor the fractions in real - time without such subsequent cumbersome identification work, reducing the workload and saving time. In the experiment, the sample vials were numbered and the fractions were collected, and at the same time, the voltage values shown on the voltmeter of the 3D metal printed UV detector were recorded for each vial when collecting the fractions, with the aim of comparing the results of the 3D metal printed UV detector with the results of ordinary column chromatography experiments.

[0076] The specific method is as follows:

[0077] 1. Column Chromatography Separation of the Salicylic Acid and Aspirin Mixed Solution:

[0078] Preparation of eluent: Mix petroleum ether, ethyl acetate and glacial acetic acid in a ratio of 50:10:1. Prepare the sample first, weigh 0.0493g of salicylic acid and 0.0535g of aspirin in a small beaker, add 3mL of 95% ethanol to dissolve, then add 1g of silica gel and mix well, heat the outer wall of the beaker with a hair dryer, and wait for 95% ethanol to evaporate. Fill the column by wet column packing, weigh 15.7g of silica gel (100-140 mesh), add an appropriate amount of eluent to stir, and place it in an ultrasonic instrument for ultrasonic degassing to prevent cracks in the column due to bubbles when the eluent is added after loading. Add 1 / 3 of the eluent to the chromatography column, add silica gel to the chromatography column while stirring, open the piston at the lower end at the same time, adjust the flow rate to 1 drop / second, and tap the outer wall of the chromatography column with an ear wash ball to form a plane with silica gel. Then connect the 3D metal printed UV detector to ensure that the flow cell in the detector is full of liquid. At this time, the value obtained by the voltmeter is the baseline. When the liquid level of the eluent drops to 1 cm above the silica gel plane, add the previously prepared sample to the chromatography column, add the eluent along the inner wall of the chromatography column for elution, and when the color band of the first component begins to flow out, collect the fractions in a vial with a volume of 2 mL, observe the value of the voltmeter at the same time and record the voltage value when each vial receives 1 mL of fraction. After the color band of the first component is completely eluted, continue eluting, and after the second component is eluted, observe whether the value of the voltmeter returns to the baseline, close the piston, and complete the elution.

[0079] 2. Identification of salicylic acid and aspirin by thin layer chromatography:

[0080] Preparation of developing agent: Mix petroleum ether, ethyl acetate and glacial acetic acid in a ratio of 30:10:1. Preparation of standard: Weigh 0.0100g of salicylic acid and dissolve it in 3mL of 95% ethanol to obtain salicylic acid standard solution; weigh 0.0102g of aspirin and dissolve it in 3mL of 95% ethanol to obtain aspirin standard solution. Perform TLC detection on the fractions collected from the chromatography column. Take the salicylic acid standard and aspirin standard as controls and use a spotting capillary to take the fractions from each vial and spot them on a GF254 silica gel plate. Place the silica gel plate in a chromatography cylinder containing the developing agent. When the solvent front is 1cm away from the upper edge of the silica gel plate, take out the silica gel plate, evaporate the developing agent, and observe it under a UV lamp to depict the positions of the fraction points and the standard points. Compare with the positions of the standard points to determine the components contained in the fraction.

[0081] 3. Identification of salicylic acid by HPLC:

[0082] A Shimadzu ODS-3-C18 liquid chromatography column (4.6 mm × 150 mm, 5 μm) was used, and the mobile phase was 0.2% phosphoric acid aqueous solution-acetonitrile (V (0.2% H 3 PO 4 Aqueous solution): V(CH3 (CN)=79:21), flow rate was 1.0 mL / min, and the detection wavelength was 254 nm.

[0083] Figure 6 It is a flowchart for the real-time monitoring of the column chromatography separation fractions of a mixed solution of salicylic acid and aspirin using a 3D metal-printed UV detector. Figure 7 In A, it shows the change curve of voltage under the real-time detection of the 3D metal-printed UV detector. From the molecular structures of salicylic acid and aspirin, it can be analyzed that the molecular structure of salicylic acid contains hydroxyl and carboxyl groups, and the intramolecular hydrogen bond greatly reduces its polarity. While aspirin contains a carboxyl group and an ester structure, and the presence of the acetyl group increases the molecular polarity. So, the polarity of salicylic acid is smaller, and the polarity of aspirin is larger. When using petroleum ether, ethyl acetate, and glacial acetic acid as eluents, salicylic acid is eluted first, and then aspirin. Therefore, it is judged that the first inverted peak appearing on the monitoring curve is salicylic acid, and the second inverted peak is aspirin. Figure 7 In B, some collected fractions were selected for TLC detection, and the results were basically the same as those of the substances detected by the 3D metal-printed UV detector. But in Figure 7 the curve marked (Ⅰ) in Figure A, in the detection of the 3D metal-printed UV detector, the voltage value has not reached the baseline voltage value, and it is considered that there are still substances with UV absorption in the fraction, namely salicylic acid. However, from the results of thin-layer chromatography, there are no corresponding spots or the spots are very light on the silica gel plate (corresponding to Figure 7 Figure C (I) in it), even when increasing the sample loading amount during spotting, it is very difficult to judge whether there is the target substance, so it is judged that there is no such target substance in the fraction. Therefore, under the condition of a wavelength of 254 nm by high-performance liquid chromatography, it is judged whether there is a target substance in the fraction. Using an aqueous solution of 0.2% phosphoric acid - acetonitrile (V(0.2% H 3 PO 4 aqueous solution): V(CH 3 CN)=79:21) as the mobile phase, and the flow rate was 1.0 mL / min. Compared with the retention time of the salicylic acid standard product ( Figure 7 Figure D in it), a small peak appears in the fraction at about a retention time of 7.2 min, as shown in Figure 7 Figure C, indicating the presence of salicylic acid in the fraction. It is very difficult to judge whether there is a target sample in the fraction by thin-layer chromatography, while in the real-time detection of the 3D metal-printed UV detector, the presence of a small amount of light-absorbing substances can be detected, with good sensitivity. This characteristic can improve the recovery rate of substances when subsequently monitoring the separation and recovery of some substances with UV absorption characteristics and low contents by the 3D metal-printed UV detector.

Claims

1. A 3D metal printed UV detector, characterized in that: The invention comprises an ultraviolet light source (1), a circulation pool (2), a chromatography column interface (3), a fraction collection port (4), a photoelectric signal conversion component (5) and a signal processing component (6); the top of the circulation pool (2) is provided with a liquid inlet (21), the bottom of the circulation pool is provided with a liquid outflow port (22), both sides of the circulation pool (2) are provided with light through ports and embedded with quartz glass sheets (23); the chromatography column interface (3) is connected to the liquid inlet (21) of the circulation pool, and the liquid outflow port (22) of the circulation pool is connected to the fraction collection port (4); the ultraviolet light source (1) and the photoelectric signal conversion component (5) are respectively arranged on both sides of the circulation pool (2); the ultraviolet light source (1) is used to emit ultraviolet light, the ultraviolet light passes through the quartz glass sheets (23) on both sides of the circulation pool (2) and is received by the photoelectric signal conversion component (5), and the photoelectric signal conversion component (5) converts the light signal into an electrical signal after receiving the light signal; the signal processing component (6) is connected to the photoelectric signal conversion component (5) and is used to receive and record the output electrical signal data.

2. The 3D metal printed UV detector according to claim 1, characterized in that: The ultraviolet light source (1) emits ultraviolet light with a wavelength in the range of 250 to 260 nm.

3. The 3D metal printed UV detector according to claim 1, characterized in that: The volume of the circulation pool (2) is 12 to 48 mm 3 .

4. The 3D metal printed UV detector according to claim 1, characterized in that: The ultraviolet light source (1) and the circulation pool (2) are connected via a fixture, and the fixture is used to fix the distance between the ultraviolet light source and the circulation pool.

5. The 3D metal printed UV detector according to claim 1, characterized in that: The photoelectric signal conversion component (5) is a silicon photocell.

6. The 3D metal printed UV detector according to claim 1, characterized in that: The signal processing component (6) is a voltmeter and an intelligent device connected thereto.

7. The 3D metal printed UV detector according to claim 1, characterized in that: A heat dissipation component is fixed on the ultraviolet light source (1).

8. The 3D metal printed UV detector according to claim 7, characterized in that: The heat dissipation component is selected from any one or more of a cooling fin, a heat sink or a heat dissipation fan.

9. Use of the 3D metal printed UV detector according to any one of claims 1 to 8 in real-time monitoring of column chromatography fraction collection.

10. The use according to claim 9, characterized in that: The application is real-time monitoring of the elution of a colorless substance with ultraviolet absorption characteristics during column chromatography.

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