CN100384500C - Quaternary low pressure gradient mixing device - Google Patents

Abstract

A quaternary low-pressure gradient mixing device in the field of biochemical analysis instruments, including a microprocessor, I/O expansion, liquid crystal display circuit, keyboard circuit, memory with clock, programmable timing circuit, solenoid valve control circuit, stirring rod control circuit , Photoelectric isolation circuit, USB communication interface circuit and control module. With the microprocessor as the core, memory with clock, solenoid valve control circuit, switching power supply, stirring rod control circuit, programmable timing circuit, I/O expansion, USB communication module are connected to it, and the solenoid valve control circuit is driven by photoelectric isolation. The circuit controls the solenoid valve, the switching power supply controls the power conversion circuit, the stirring rod control circuit controls the stirring rod through the photoelectric isolation drive circuit, the P89C668 microprocessor controls the liquid crystal display circuit and the keyboard circuit through the I/O expansion, and the P89C668 communicates with the computer through the USB communication module. connect. The elution method adopted in the present invention greatly reduces the cost, and has strong applicability and economy.

Description

Quaternary low pressure gradient mixing device

technical field

The invention relates to a device in the field of biochemical analysis instruments, in particular to a quaternary low-pressure gradient mixing device used in high-performance liquid chromatography.

Background technique

The high-performance liquid chromatograph controlled by a microprocessor now has a high degree of automation. It can not only control the operating parameters of the instrument, but also shrink, enlarge, and superimpose the obtained chromatograms, as well as retain data and peak heights and peaks. It provides high-efficiency and comprehensive analysis tools for chromatographers. The gradient precipitation system in high performance liquid chromatography is divided into high pressure gradient and low pressure gradient. There are generally two types of high-pressure gradient devices, one is composed of two high-pressure pumps, a gradient program controller, a mixer and other components. This high-pressure gradient device can obtain any form of gradient curve as long as the program controls the output of each pump, and has high precision and is easy to realize automatic control. Its main disadvantage is that it uses two high-pressure pumps, which are relatively expensive and have a high probability of failure. And limited to the mixture of two solvents. Another high-pressure gradient device uses only one high-pressure pump, and often uses a constant-pressure pump to cooperate with a program-controlled solenoid valve to realize gradient supply. The advantage of this device is that only one high-pressure pump is used, but two high-pressure solenoid valves are required. The low-pressure gradient device is relatively simple. The solvents are first mixed in a certain proportion under low pressure or gravity, and then input into the chromatographic column by a high-pressure pump. The low-pressure gradient can mix more than two solvents, and the mixing system does not need a high-pressure valve, and only uses a high-pressure pump to realize the gradient infusion. Solvent mixing is often accompanied by volume changes due to differences in viscosity, density, and compressibility, which are an important source of some deviations in gradient repeatability. Theoretically, low-pressure gradients are more reproducible than high-pressure gradients because solvents are mixed before pressurization. Modern low-pressure gradient devices use automatic switching valves controlled by programmable microcontrollers. Solvent mixing is controlled by a programmable microcontroller to automatically switch valves, so that each solvent storage tank path is sequentially connected at a certain time interval. A linear gradient can be obtained when the switching valve sequentially connects the solvent passages at equal time intervals. If the time for switching the switching valve to connect each solvent passage is long at the beginning and then short or short at the beginning and then long, different exponential gradients or logarithmic gradients can be obtained. Compared with the double-pump high-pressure gradient system, the single-pump low-pressure gradient system is greatly simplified, and the failure rate of the instrument is low. Moreover, the volume change caused by solvent mixing can be reduced, the gradient repeatability is good, and the precision is high.

According to the literature search of the existing technology, it is found that there is no self-developed low-pressure gradient mixing device in my country, and it mainly relies on foreign imports. Japanese Patent No. 58079155 and U.S. Patent No. 4311586 respectively describe a gradient mixing device for liquid chromatography, wherein they both use two low-pressure pumps to mix two solvents in a certain proportion, and then pass through a high-pressure pump to realize gradient infusion. The main defect of this design is that the structure is complicated and the cost is too high.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention proposes a quaternary low-pressure gradient mixing device used in high-performance liquid chromatographs, so that it can fill the domestic blank, and can greatly reduce the cost of high-performance liquid chromatographs at the same time. Strong applicability and economy.

The present invention is achieved through the following technical solutions, the present invention comprises: microprocessor (MCU), I/O expansion, liquid crystal display circuit, keyboard circuit, memory with clock, programmable timing circuit, solenoid valve control circuit, stirring bar Control circuit, photoelectric isolation driving circuit, USB communication module and control module. Among them, the microprocessor is the core, and the memory with clock, solenoid valve control circuit, switching power supply, stirring rod control circuit, programmable timing circuit, I/O expansion, USB communication module are connected to it. The drive circuit controls the solenoid valve, the switching power supply controls the power conversion circuit, the power conversion circuit is used to control the photoelectric isolation drive circuit, the stirring rod control circuit controls the stirring rod through the photoelectric isolation drive circuit, and the microprocessor controls the liquid crystal display circuit and The keyboard circuit, the microprocessor is connected with the computer through the USB communication module. The control module realizes the adjustment and control of the hardware so as to complete the gradient elution task.

The microprocessor controls the liquid crystal display output and keyboard information input through the I/O expansion port, completes the channel selection, parameter setting, and converts the parameter into the switching time of the time proportional solenoid valve through the algorithm and the switching time of the solenoid valve obtained according to the algorithm The solenoid valve and the stirring rod are controlled by the solenoid valve control circuit and the stirring rod control circuit, thereby completing the gradient elution. The memory with a clock saves the parameters set by the low-pressure gradient elution to facilitate repeated experiments by the user, and also stores the data for controlling the time-proportional solenoid valve. The programmable timing circuit is mainly used to control the switching time of the time proportional solenoid valve. The USB communication module realizes the data transmission between the PC and the present invention.

The control module of the present invention is a system control application program written in C language, and the system control mainly realizes system initialization, parameter setting, two-way communication with the PC of the chromatographic data workstation through a USB interface, obtaining the gradient data of the elution curve through parameters, Carry out gradient elution (that is, control the opening and closing of the time proportional solenoid valve according to the elution curve), display real-time refresh and input and output control.

The system control application program in the control module of the present invention is divided into a main program and several interrupt service programs, and tasks with low real-time requirements are placed in the main program, and are executed sequentially until the end of the gradient elution, and then wait for the next gradient elution; Place tasks with high real-time requirements (such as controlling the switching off of solenoid valves and identifying keys) in the interrupt service routine for timely processing.

In order to improve the reproducibility of the gradient transfusion, the invention uses a microcomputer control system to compile and control the program of the solvent gradient. The preparation method of the solvent gradient program is to store the preprogrammed gradient program curve in the memory of the controller, and there are straight line, concave and convex gradient curves. Five kinds of convex curves and five kinds of concave curves were used in the establishment of the gradient elution data module, and the continuously changing gradient elution curves were converted into discrete gradient elution curves, so that the microprocessor could control the opening and closing of the solenoid valve.

The microprocessor of the present invention adopts the P89C668 single-chip microcomputer, takes the P89C668 single-chip microcomputer as the control core, and uses the microcomputer control to continuously change the composition of the mobile phase in the gradient elution, to adjust the polarity of the mobile phase, so that each component that flows out has a suitable Capacity factor k', and all the components in the sample can be separated satisfactorily and selectively at the applicable resolution within the shortest analysis time.

The invention adopts the low-pressure gradient elution method, the low-pressure gradient system is greatly simplified compared with the high-pressure gradient system, and the failure rate of the instrument is low. Moreover, the volume change caused by solvent mixing can be reduced, the gradient repeatability is good, and the precision is high. Moreover, the cost is greatly reduced, and the utility model has strong applicability and economy.

The invention adopts a solvent gradient programming method in which the solvent gradient program is pre-stored in the memory of the controller. The procedures for programming are simplified, the reproducibility of gradient infusion is improved, and it can be applied to various gradient elutions.

Description of drawings

Fig. 1 is a block diagram of the hardware system of the present invention.

Fig. 2 is the interface circuit between liquid crystal module M19264-2A3 and microprocessor P89C668 in the present invention.

Fig. 3 is the interface circuit of programmable timer 8253 and microprocessor P89C668 in the present invention.

Fig. 4 is the electromagnetic valve control circuit in the present invention.

Fig. 5 is the stirring bar control circuit in the present invention.

Fig. 6 is the interface circuit of USB interface chip FT245BM and microprocessor P89C668 among the present invention.

Figure 7-1 is the connection diagram of LT1086-12 in the power supply design circuit of the present invention.

Figure 7-2 shows the +12V to +5V power supply realized by LT1129 in the power supply design circuit of the present invention.

Fig. 8 is a flow chart of the main program of the control module in the present invention.

Fig. 9-1 is a flowchart of the elution module in the gradient elution module in the present invention.

Fig. 9-2 is a flowchart of timer T0 interruption in the gradient elution module of the present invention.

Detailed ways

The invention proposes a low-pressure gradient elution device used in a high-performance liquid chromatograph, which is realized by two parts of hardware and control, and the hardware part can be realized by using the prior art. In conjunction with each accompanying drawing, describe in detail as follows:

As shown in Figure 1. The low pressure gradient mixing device of the present embodiment is composed of a microprocessor (MCU), I/O expansion, liquid crystal display circuit, keyboard circuit, memory with clock, programmable timing circuit, electromagnetic valve control circuit, stirring bar control circuit, photoelectric Isolated driving circuit, USB communication module and control module.

In this embodiment, the microprocessor part is the core of the low-pressure gradient mixing device. It adopts Philips' P89C668 as the CPU of the system. The switching time of the valve and the switching time of the solenoid valve obtained by the algorithm are used to control the solenoid valve, thereby completing the gradient elution.

In this embodiment, a programmable parallel interface 8255 is used as an I/O expansion for external connection of a liquid crystal display and a matrix keyboard.

In this embodiment, the M19264-2A3 liquid crystal display of TRULY Company is adopted, which can display 192 characters and 48 Chinese characters. The liquid crystal module is connected with the microprocessor, and the display mode and other parameters are set by using special liquid crystal instructions to realize the display of the control and processing information of the low-pressure gradient mixing device. The interface circuit between M19264-2A3 LCD module and P89C668 is shown in Figure 2.

In this embodiment, a keyboard with ten number keys from 0 to 9, a decimal point key and function selection and parameter setting keys including channel selection, parameter setting, run/stop and cursor movement are used. The control of the keyboard is completed by the programmable expansion parallel interface 8255A.

In this embodiment, a programmable timing circuit is used to control the switching time of the time proportional solenoid valve. The timing circuit adopts Intel programmable timer/counter interface circuit 8235 to realize. The control circuit connected with the microprocessor is shown in Figure 3.

In this embodiment, the P1 port of the single-chip microcomputer is used to drive the Panasonic solid-state relay (GUPhotoMOS AQW212, which is a double-group solid-state relay) through the driver 75451 to directly control the switch of the solenoid valve, thereby forming a control circuit for the solenoid valve. As shown in Figure 4. Take solenoid valve 1 as an example, when P1.7 outputs a high level, the corresponding 75451 also outputs a high level, so the LED between pin 1 and pin 2 of the AQW212 does not emit light, so the MOSFET does not conduct and the load is disconnected. That is, the solenoid valve is closed; when P1.7 outputs low level, the corresponding 75451 also outputs low level, so the LED between pin 1 and pin 2 of AQW212 lights up, so that the MOSFET is turned on, and the load is turned on, that is, the solenoid valve is opened . In this way, through the timing circuit, controlling the time when the P1.7 port maintains high and low levels realizes the control of the on-off time of the solenoid valve, thereby realizing the mixing of gradient elution solvents.

In this embodiment, the principle of the stirring rod control circuit is the same as that of the solenoid valve control circuit, and the difference has the following two points: First, the solid state relay used in the solenoid valve control circuit is GU PhotoMOS AQW212, and the solid state relay used in the stirring rod control circuit It is POWER PhotoMOS AQZ102; Second, the solenoid valve control circuit is directly controlled by the P1.4-P1.7 port, and the stirring rod control circuit is controlled by the PC2 port of the 8255 to control the GATE1 port of the 8253, and then controlled by the square wave output by OUT1 Stirring rod, the reason is that the stirring speed of the stirring rod is too fast to easily wear the inner wall, and the stirring speed does not need to be too fast. The control circuit of the stirring rod is shown in Figure 5.

The low-pressure gradient mixing device in this embodiment uses a USB interface to communicate with a PC to form a chromatographic data workstation. In this embodiment, the USB interface chip FT245BM of FTDI Company is used to connect with the microprocessor P89C668, and its hardware interface circuit is shown in Fig. 6 . The FT245BM is directly connected to the P89C668, the data port of the FT245BM is directly connected to the data bus of the P89C668, the RD and WR of the FT245BM are directly connected to the RD and WR of the P89C668, and the two P1 ports of the P89C668 are used to identify the handshake signal of the FT245BM ( RXF, TXE). When TXE# is low, it means that the current FIFO sending buffer is empty, and data can be written to USB. At this time, when the WR pulse changes from high to low, the data on D0~D7 will be written into the FIFO sending buffer area; when TXE #When it becomes high, it means that the current FIFO sending buffer is full or is storing the last byte, and it is forbidden to write data to the sending buffer; when RXF# is low, it means that the current FIFO receiving buffer has data, at this time the RD pulse is sent by When low becomes high, data will be read from the FIFO receiving buffer; when the read signal RD is low, the data will be read to the data lines D0~D7; when RXF# is high, reading data from the FIFO receiving buffer is prohibited.

In this embodiment, +5V, isolated +5V and +24V power sources are used during operation, and the AC 220V power source needs to be converted into the required power source. In this embodiment, MEAN WELL switching power supply D-50B (GD) is used to isolate and convert to +5V and +24V power supplies that do not share a common ground. In this way, it is necessary to convert the +24V power supply to isolated +5V, so the low-voltage The dropout linear regulator LT1086CT-12 module converts the +24V supply to +12V, and generates an isolated +5V supply through the low dropout linear regulator LT1129CQ. As shown in Figure 7.

The control module of this embodiment is a system control application program written in C language, which mainly realizes system initialization, parameter setting, two-way communication with the PC of the chromatographic data workstation through the USB interface, obtaining gradient data of the elution curve through parameters, and performing Gradient elution (that is, control the opening and closing of the time proportional solenoid valve according to the elution curve), display real-time refresh and input and output control.

The flowchart of the main program in the control module in this embodiment is shown in FIG. 8 . The specific steps are as follows:

1. start;

2. System initialization;

3. Display the boot screen;

4. Determine whether there is a key input;

5. If there is key input, go to 6; if there is no key input, go to 4;

6. Determine whether it is the "local/network" button;

7. If it is the "Local/Network" key, reverse the network sign;

8. Determine whether it is connected to the Internet;

9. If it is in the networked state, set the parameters through the chromatographic workstation to 16; if it is not in the networked state, judge whether to press the "channel selection" button;

10. If you press the "Channel Selection" button, call the channel selection subroutine; if you don't press the "Channel Selection" button, go to 4;

11. Determine whether there is a key input;

12. If there is key input, go to 13; if there is no key input, go to 11;

13. Determine whether it is the "parameter setting" key;

14. If you press the "Parameter Setting" button, go to 15; if you don't press the "Parameter Setting" button, go to 11;

15. Call parameter This is the key subroutine;

16. Establish gradient elution data;

17. Determine whether there is a key input;

18. If there is a key input, then go to 19; if there is no key input, go to 17;

19. Determine whether it is the "Start" key;

20. If it is the "Start" key, go to 21; if it is not the "Start" key, go to 3;

21. Perform gradient elution.

In this embodiment, the control module follows a modular design method, and the establishment of the gradient elution data module and the gradient elution module are the keys of the main program. To establish the gradient elution data module, the application program automatically activates the corresponding subroutine after the parameter setting is completed; for the gradient elution module, the user activates the corresponding subroutine through the "run/stop key" after the gradient elution data is established , There are two ways to end the gradient elution: the system automatically ends the elution time and the user forces the end through the "run/stop key". Each module of the main program is introduced separately below.

The system initialization in this embodiment includes interrupt-related register setting, interrupt priority, initialization of the programmable parallel interface 8255A, and liquid crystal display screen clearing.

In this embodiment, 5 kinds of convex curves and 5 kinds of concave curves are used in the establishment of the gradient elution data module, and the continuously changing gradient elution curves are converted into discrete gradient elution curves, so that the microprocessor can control the time proportional electromagnetic valve disconnection.

In this embodiment, after the gradient elution data is established, the gradient elution module controls the opening and closing of the time proportional solenoid valve to realize the gradient elution. The flow chart of the elution module is shown in Figure 9-1. Specific steps are as follows:

1. start;

2. Set the mixing cycle according to the flow;

3. Take the initial concentration ratio of gradient elution;

4. Take the first mixing cycle number;

5. Start timer 0;

6. Open the main solenoid valve;

7. The main solenoid valve open flag is set to 1;

8. Start the stir bar

9. Call the display subroutine;

10. Delay for 1 second;

11. Determine whether there is an end key pressed;

12. If so, go to 15; if not, go to 13;

13. Determine whether the experiment time is up;

14. If the experiment time is up, go to 15; if the experiment time is not up, refresh the display cache, go to 9;

15. Close the solenoid valve;

16. Close the stir bar;

17. Turn off the timer;

18. Return.

The timer T0 interrupt flow chart is shown in Figure 9-2, and the specific steps are as follows:

1. start;

2. Determine whether the open flag of the main solenoid valve is 1;

3. If yes, go to 4; if not, go to 14;

4. Open the secondary solenoid valve and close the main solenoid valve;

5. The number of mixing cycles is reduced by 1;

6. Determine whether the number of mixing cycles is 0;

7. If it is 0, go to 8; if not 0, go to 10;

8. Refresh the concentration ratio in the display buffer;

9. Refresh the number of mixing cycles;

10. Assign an initial value to the timer;

11. Start timer 0;

12. Open timer 0 interrupt;

13. Return.

14. Open the main solenoid valve and close the secondary solenoid valve;

15. Assign the initial value to timer 0 to 11.

The key-value processing module in this embodiment adopts an interrupt method, reads the key value in the interrupt service program, and stores it in the key-value buffer, and performs corresponding processing on the key value in the main program.

In this embodiment, the liquid crystal display module cooperates with the keyboard to complete the man-machine dialogue (including the setting of various parameters, real-time display of the proportion of each channel during the gradient elution process, the remaining time of elution, and the elution curve used, etc.).

The USB communication module in this embodiment is to realize the data transmission between the PC and the embedded system with the USB interface.

Claims (4)

1. quarternary low pressure gradient mixing device, comprise: microprocessor, the I/O expansion, liquid crystal display circuit, keyboard circuit, the memory of band clock, programmable timing electric circuit, solenoid valve control circuit, the stirring rod control circuit, photoelectric isolating driving circuit, usb communication module and control module, it is characterized in that, with the microprocessor is core, the memory of connecting band clock on it, solenoid valve control circuit, Switching Power Supply, the stirring rod control circuit, programmable timing electric circuit, the I/O expansion, the usb communication module, solenoid valve control circuit is controlled magnetic valve by photoelectric isolating driving circuit, Switching Power Supply control power-switching circuit, power-switching circuit is used to control photoelectric isolating driving circuit, the stirring rod control circuit is controlled stirring rod by photoelectric isolating driving circuit, microprocessor is by I/O expansion control liquid crystal display circuit and keyboard circuit, microprocessor interrelates by usb communication module and computer, control module realizes adjusting and the control to hardware, so that finish the gradient elution task;

Described control module is the system's controlling application program with the C language compilation, realize system initialization, parameter setting, with the PC of chromatographic data work station by USB interface carry out two-way communication, by parameter obtain elution curve gradient data, carry out gradient elution, show and refresh in real time and input and output control;

System's controlling application program in the described control module is divided into a main program and several interrupt service routine, places real-time in the main program and requires low task, and order is carried out up to gradient elution and finished, and waits for gradient elution next time then; Place the demanding task of real-time in the interrupt service routine, comprise the identification of the cut-offfing of control magnetic valve, button, so that in time handle;

The program of solvent gradient is worked out and is controlled by Control System of Microcomputer, the preparation method of solvent gradient program is to store the gradient program curve that prelists in the controller internal memory, the gradient curve that straight line, spill, convex are arranged, set up and adopted 5 kinds of crest curves and 5 kinds of concave curves in the gradient elution data module, continually varying gradient elution curve is converted to discrete gradient elution curve, so that the cut-offfing of microprocessor controls solenoid valves.

2. quarternary low pressure gradient mixing device as claimed in claim 1, it is characterized in that, microprocessor is by I/O expansion mouthful control liquid crystal display output and keypad information input, finish selection, the parameter of passage setting, be converted to the switch time of magnetic valve by algorithm and according to going to control magnetic valve and stirring rod by solenoid valve control circuit and stirring rod control circuit the switch time of the magnetic valve of algorithm gained according to parameter, thereby finish gradient elution.

3. quarternary low pressure gradient mixing device as claimed in claim 1 is characterized in that, the memory of band clock is preserved the set parameter of low pressure gradient wash-out, carries out repeated test to make things convenient for the user, also will control the data of magnetic valve simultaneously and also store wherein.

4. quarternary low pressure gradient mixing device as claimed in claim 1 is characterized in that programmable timing electric circuit is used for controlling the switch time of magnetic valve.

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