There are still many applications of HPLC in our life. Its system consists of reservoirs, pumps, injectors, columns, detectors, and recorders. The mobile phase in the reservoir is pumped into the system by the high pressure pump. The sample solution enters the mobile phase through the sampler and is loaded into the column (stationary phase) by the mobile phase. Since the components in the sample solution have two phases, Different distribution coefficients, when used for relative motion in two phases, undergo repeated adsorption-desorption distribution processes, and each component has a large difference in moving speed, and is separated into individual components and sequentially flowed out of the column. When passing through the detector, the sample concentration is converted into an electrical signal and sent to the recorder. The data is printed out as a map.

1 System composition and working principle of HPLC system 2 Application of HPLC The HPLC method only requires the sample to be made into a solution that is not limited by the volatility of the sample. The mobile phase may have a wide range of options and may be fixed. The variety of phases allows the separation of thermally unstable and non-volatile, dissociated and non-dissociated and various molecular weight ranges.

In combination with sample pretreatment technology, the high resolution and high sensitivity achieved by HPLC make it possible to separate and simultaneously determine substances that are very similar in properties and to separate trace components in complex phases. With the development of the stationary phase, it is possible to complete its separation while maintaining the activity of biochemical substances.

HPLC has become the most promising method for solving biochemical analysis problems. Because HPLC has the advantages of high resolution, high sensitivity, high speed, the column can be repeatedly used, and the outflow components are easily collected, it is widely used in various fields such as biochemistry, food analysis, pharmaceutical research, environmental analysis, inorganic analysis, etc. . The combination of HPLC and structural instruments is an important development direction.

Liquid chromatography-mass spectrometry technology has received widespread attention, such as the analysis of carbamate pesticides and polynuclear aromatics; liquid chromatography-infrared spectroscopy has also developed rapidly, such as the determination of hydrocarbons in water in environmental pollution analysis, not in seawater Volatile hydrocarbons have made new developments in environmental pollution analysis.

3 High Performance Liquid Chromatograph Common Troubleshooting The LC-10AT high performance liquid chromatograph was manufactured by Shimadzu Corporation in 1996. The detector was a variable wavelength UV detector. The column was ODS-C18 of Shimadzu Corporation. Special ODS-C18.

During the eight years of daily work, several kinds of failures were encountered. For example, failure 1 was resolved after consulting the engineers of Shimadzu Corporation. Fault 2 and fault 5 were frequently encountered problems. The following are summarized as follows:

Fault 1 There is a bubble in the mobile phase. Turn off the pump, open the pressure relief valve, open the purge key, and clean the degassing. The air bubbles continuously emerge from the filter and enter the mobile phase. Whether you open the purge key several times, you cannot clear the continuous air bubbles. .

Causes The filter is immersed in buffers such as ammonium acetate for a long time. The interior of the filter grows and proliferates due to mold growth, blocking the filter, making it difficult for the buffer to smoothly pass through the filter. Air enters the filter under pressure from the pump. Mobile phase.

The filter is soaked in a 5% nitric acid solution and washed ultrasonically for a few minutes. The filter may be soaked in a 5% nitric acid solution for 12 to 36 hours. The filter is gently shaken several times, and the filter is washed several times with pure water. , Open the pressure relief valve, open the purge key, clean the degassing. If bubbles continue to emerge from the filter, continue immersing the filter in a 5% nitric acid solution. If no air bubbles continuously emerge from the filter, indicate the filter. The mold inside the mold has been destroyed by nitric acid and the mobile phase can pass through the filter smoothly. Open the pressure relief valve, open the pump, adjust the flow rate to 1.0 to 3.0 ml/min, and rinse the filter with pure water for about 1 hour. The filter can be cleaned. Close the pressure relief valve and pure methanol rinse for half an hour.

Failure 2 column pressure reasons (1) buffer salt such as (ammonium acetate, etc.) deposited in the column; (2) sample pollution deposition.

For the first case, first use the pure water of 40-50°C, and flush the column forward at a low speed. After the column pressure is gradually decreased, flush the flow rate accordingly. After the column pressure drops sharply, rinse with normal temperature pure water, and then use pure water. The column was flushed with methanol for 30 minutes; for the second case, a contaminated C18 column caused by the deposition of the sample was flushed back with pure water and then flushed with methanol, followed by washing with methanol isopropanol (46) (flushing The length of time depends on the contamination of the sample), rinse with methanol, rinse with pure water, and rinse the column for 30 minutes.

Fault 3 has neither pressure indication nor liquid flow [1].

Causes (1) Pump packing wear; (2) Large amount of air bubbles enter the pump body.

Handling For the first case, replace the sealing gasket; for the second case, use a 50-ml glass syringe to assist with air extraction at the pump outlet while the pump is working.

Fault 4 pressure fluctuations, flow instability.

Cause There is air or foreign matter in the system between the jewel ball and the valve seat of the one-way valve, so that the two cannot be sealed.

Observe the amount of mobile phase in the processing work to ensure that the stainless steel filter sinks into the bottom of the accumulator and avoid inhaling air. The mobile phase should be fully degassed [2]. If there is a foreign matter between the check valve and the valve seat, remove the check valve and place it in a beaker containing acetone to clean it with ultrasonics.

Fault 5 is out of peak, peak split.

Causes (1) The column was contaminated; (2) The column head was collapsed.

Treatment For the first case, the column was first flushed with pure water, then flushed with methanol. The column was then washed with methanol isopropanol (46) (the length of the rinse time was determined by the contamination of the sample). Rinse with methanol and then rinse with pure water. Finally rinse the column with methanol for more than 30 minutes. If the peak after washing is still poor, consider the second case. For the second case, unscrew the stigma and check the column packing for induration or collapse. Remove the indurated part (contaminated filler), fill the new filler, drop a drop of methanol, fill the filler, refill it, press it with a stainless steel rod with the same diameter as the top of the column, fill it with flat, drop the methanol, and then compress it repeatedly. Times until full fill [2>. The stigma is rinsed with methanol, wipe the packing on the outer wall of the column, tighten the stigma, and rinse with pure methanol for more than 30 minutes.

Fault 6 has poor peak area repeatability.

Causes (1) Injection valve leaks; (2) Needle is not in place.

Handling Replace the injection valve gasket for the first case; for the second case ensure that the pipetting needle is inserted completely, and the sample solution must be quickly and smoothly transferred from the LOAD state to the INJECT state after injection of the sample solution to ensure accurate injection volume. In daily work, the maintenance of the liquid chromatograph is very important, for instance, should pay attention not to allow the air to enter the infusion system and the high-pressure pump, the solution in the reservoir should not be used for a long time to clean the reservoir and replace the solution, each time After the chromatograph, the buffer should be rinsed with pure water to prevent precipitation or deposition of inorganic salts. Pre-treatment of the sample is also very important. Any sample should be as much as possible to remove impurities, completely dissolved, to minimize the column pollution. Extend the service life of the column, while avoiding the injection of concentrated sample solution, so as to prevent the solid solution from blocking due to precipitation of the residual liquid in the injection valve. The column should be marked and the column used for different analysis purposes should not be mixed.