As a precision analytical instrument, the performance stability and service life of high-performance liquid chromatography are highly dependent on scientific maintenance. The following systematically elaborates on the maintenance points of HPLC from the aspects of operating specifications, component maintenance, environmental control, and fault handling, to help users avoid common problems and extend the service life of the instrument.

1、 Daily maintenance: preventive operation is key

1. Maintenance of infusion system

-Mobile phase management:

① The configuration of the mobile phase requires the use of ultrapure water (resistivity ≥ 18.2 M Ω· cm) or chromatographically pure solvent, filtered through a 0.45 μ m membrane and ultrasonically degassed for 15 minutes to avoid bubble or particle blockage of the system.

② Rinse the pipeline with isopropanol or methanol before daily testing to replace residual solvents and prevent salt precipitation or microbial growth.

-Pump maintenance:

① Clean the pump head promptly after operation and prohibit corrosive solvents (such as concentrated sulfuric acid). Mechanical pumps require regular inspection of the sealing ring, and any leaks should be replaced immediately.

② Avoid dry suction (when the pump is running without liquid), which may cause wear on the pump head or damage to air pockets.

2. Cleaning of the injection system

-Automatic sampler:

① After each test, rinse the injection needle with methanol water (50:50) and wipe off any residue outside the needle with lens paper.

② Disassemble the injection valve weekly and immerse it in a 10% nitric acid solution for ultrasonic cleaning to remove buffer salt crystals.

-Manual injection:

Ensure sample injection to avoid residual contamination of the quantitative loop. High viscosity samples need to be diluted before sampling.

3. Column protection

-Operating standards:

① It is strictly prohibited to directly inject unfiltered samples. It is recommended to pre install a 0.22 μ m online filter.

② Avoid overpressure operation (usually not exceeding 80% of the nominal pressure of the column), as sudden high pressure may cause the column bed to collapse.

- 保存方法：

The reverse phase column (C18) needs to be stored in methanol water (1:1), the normal phase column (silica gel column) should be sealed with n-hexane, and a preservative (such as 0.05% sodium azide) should be added to the biochemical column (such as protein column).

4. Detector maintenance

-UV detector:

① Regularly wipe the flow cell window with ethanol to prevent sample residue from causing a decrease in transmittance.

② The deuterium lamp has a lifespan of about 2000 hours and needs to be replaced when its energy decays to 70% of its initial value.

-Fluorescence detector:

Rinse the circulation pool with clean water after each test to avoid residual fluorescent substances interfering with subsequent testing.

2、 Regular deep maintenance: ensuring long-term stability

1. System flushing and calibration

-Quarterly maintenance:

① Rinse the metal pipeline with a 5% nitric acid solution to remove oxidized deposits; The reverse phase system can be cleaned with 0.1% phosphoric acid.

② Calibration of flow rate accuracy: The deviation between the actual flow rate measured by a stopwatch and the set value should be less than 5%.

-Annual maintenance:

① Replace aging pipelines (such as PEEK pipes becoming brittle or discolored).

② Verify the wavelength accuracy of the detector (calibrate the ultraviolet wavelength using a holmium glass filter).

2. Column regeneration and evaluation

-Column effect recovery:

Reverse phase columns can be backwashed with solution to remove strongly adsorbed substances; The ion exchange column needs to be regenerated with a high concentration salt solution.

-Performance evaluation:

Every six months, test the column efficiency (such as the number of uracil peak trays ≥ 80% of the theoretical value), retention time drift (<15%), and tailing factor (≤ 2.0%).

3、 Long term shutdown treatment: prevent component damage

1. Short term shutdown (<1 week):

-After rinsing the system with methanol, turn off the machine and keep the chromatography column filled with solvent.

2. Long term shutdown (>1 week):

-Disassemble the chromatographic column for storage, inject silicone oil into the pump head for rust prevention, and seal the detector flow cell after drying. Place the entire machine in a moisture-proof box (humidity<40%).

4、 Environmental Control and Operating Standards

1. Temperature and humidity management: The laboratory temperature should be controlled at 20-25 ℃, and the humidity should be less than 60% to avoid condensation water corroding optical components.

2. Dust prevention measures: The instrument should be covered with a dust cover, and the air purification system should maintain PM10<50 μ g/m ³.

3. Operation taboos: It is strictly prohibited to plug or unplug with power on. Before sudden power failure, the host power must be turned off first.

5、 Data recording and spare parts management

1. Maintenance log: Record the maintenance content, replacement part batch number, and instrument status for easy traceability.

2. Spare parts reserve: Conventional spare injection valve, sealing gasket, deuterium lamp, chromatographic column (it is recommended to backup 2 of the same model).