A set of liquid chromatography systems is roughly divided into the following sections: mobile phase pretreatment section, infusion section, injection section, separation section, and detection section.
First, the mobile phase pre-treatment
The mobile phase pretreatment mainly includes filtration and degassing, and the equipment is mainly used as a solvent filter, a vacuum pump and ultrasonic waves.
1. Filter. The mobile phase used in HPLC must be pre-filtered and degassed. The purpose of filtration is to prevent particulate impurities from clogging the system or affecting the normal operation of the check valve. Attention should be paid to the selection of the filter when filtering. The oil film and the aqueous film must be used separately. The water film is easily dissolved in the organic solvent and enters the system, causing blockage of the pump flow path and the column head.
2. Ultrasound. The role of ultrasound is to remove air bubbles from the mobile phase. The ultrasonic time is generally 20 minutes. After sonication, the temperature of the liquid will rise and should be cooled to room temperature before use. If the mobile phase after ultrasound is used, it is necessary to use ultrasound again every other day.
Second, the infusion system
The infusion system is a high-pressure constant-flow pump that provides a stable and accurate flow rate.
1. Solvent filter head. Aspirating filter heads, or sinkers, are primarily used to filter particulate impurities that may be present in the mobile phase. After prolonged use, impurities may block the pores of the filter plate on the solvent filter head; or when the buffer is used for a long time, the surface of the filter head is prone to a film that hinders the normal passage of the mobile phase. In severe cases, even if the solvent has been sonicated, air bubbles will be generated in the PTFE tube when the pump is aspirating. Therefore, the filter head should be cleaned frequently. The cleaning solvent can be selected from ethanol or 30% dilute nitric acid solution, and the normal phase system should pay attention to the drying process of the filter head.
2. One-way valve. The function of the check valve is to ensure that the liquid flows in one direction, which is the guarantee for the stable infusion of the high pressure constant current pump. During daily use, you can determine whether the flow is normal by observing the pressure. If the system has been balanced for some time, the pressure should be stable. However, if the pressure fluctuates, it indicates that the flow is unstable; if there is no pressure, it means no flow. Both of these conditions are mostly due to the inclusion of air bubbles or impurities in the check valve. When mixing air bubbles, open the vent valve and press the flush button to discharge the air bubbles inside. When the check valve is mixed with impurities, it must be cleaned. Ethanol can be selected as the cleaning solvent, and the direction of the marking ring should be noted during installation. Check valve assembly drawing and sectional view 1 marked ring valve casing; 2 ceramic seat; 3 gem ball; 4 with gasket limit sleeve; 5 gasket
3. Sealing ring. The sealing ring is fixed on the plunger rod to prevent liquid leakage in the pump chamber, and is a key component for ensuring the normal operation of the pump head. However, the long-term use of the plunger rod and the plunger seal wears, mainly related to the flow rate, operating pressure and the mobile phase used. When a salt-containing mobile phase is used, salt crystallization may form due to dehydration or evaporation, and salt crystallization may cause wear of the seal ring and the plunger rod during pumping. Therefore, it is necessary to rinse the sealing ring (between the plunger sealing ring and the secondary sealing ring) with pure water before and after the experiment every day, keep the water in the cleaning tube to prevent the formation of crystals, and extend the plunger rod and the sealing ring. Service life. Isocratic system gradient system plunger rod cleaning connection diagram
4. Online filter. In order to prevent impurities from entering the chromatographic system due to mobile phase, the pump installs an in-line filter in the vent valve, and the liquid flowing out through the pump outlet passes through the inline filter, and the liquid flowing out through the venting tube does not pass through the inline filter. After the instrument has been used for a certain period of time, it is recommended that the user clean the sintered stainless steel filter of the inline filter. Use a wrench to remove the pressure cap, take out the seal ring together with the sintered stainless steel filter, clean it, and install it in the original position. The cleaning solvent can choose 30% dilute nitric acid solution, and the normal phase system should pay attention to the drying process of the filter head.
Third, the injection system
The injection system is divided into a manual injection valve and an autosampler.
1. Manual injection valve. For the manual injection valve, it is necessary to pay attention to the injection valve after each sample is injected to prevent the residual from affecting the analysis of the next sample. Also need to pay attention to the injection method, taking the 20μL loop as an example, we can choose to fully fill the quantitative tube or partially fill the quantitative tube. In order to ensure the repeatability of the injection, when the full-filled tube injection method is selected, it is necessary to inject the sample with the volume of the loop more than 3 times; when the part is filled with the tube-injection method, the injection volume should be less than half of the volume of the tube. That is 1-10 μL.
2. Autosampler. If using the autosampler injection, first of all need to ensure that there are no bubbles in the syringe, otherwise it will affect the accuracy of the injection volume; secondly, there are enough samples in the sample bottle to ensure that the sample can suck the sample. To avoid cross-contamination, the vials, lids, and pads need to be cleaned regularly. If the autosampler is not used for a long time, it should be noted that corrosive mobile phases or lotions (for example, alkaline or acidic buffer solutions) must be completely removed from the system. At the same time, in order to avoid bacterial growth, a sample vial should be filled with methanol and repeated several injections.
Fourth, the separation system
The separation system consists of a column, a guard column, and a column oven.
1. Column. The column is the core of sample separation. The column must be read carefully before use to understand the pH range, solvent tolerance range, pressure range, and maintenance methods used for the column. Once the column is in use, flush the column in time. After the rinsing is completed, the column should be removed from the instrument, sealed at the ends with the manufacturer's plug, and stored in the column box.
2. Protection column. The role of the guard column is to prevent contamination of the column by highly adsorbable impurities, thereby extending the life of the column. For different types of columns, the guard column for the corresponding packing should be selected. It should be noted that the core is also long-lived and should be replaced periodically.
3, column temperature box. Changes in column temperature may result in changes in retention time. To avoid this problem, it is recommended to use a column oven.
Five, detection system
A commonly used detector for HPLC is a UV detector. In the daily use process, the UV detector mainly needs to pay attention to the use and maintenance of the detection tank and the xenon lamp.
1. Detection pool. Long-term use of the test cell may cause pollution. If the reference energy (REF ENERGY) of the instrument is normal and the measured energy (SMP ENERGY) is low, it may be caused by contamination of the test cell, and the test cell may be cleaned. To clean the parts of the test cell, firstly, ultrasonic cleaning is performed with a 1:4 nitric acid solution, and then washed with pure water and methanol solution respectively, then reassembled and the test cell body is pushed into the cell cavity, and the plate plate screw is tightened. Note that in the assembly, the pool glass and gasket must be placed in order to avoid crushing the pool glass and causing the detection tank to leak.
2. Xenon lights. The normal life of the xenon lamp can be more than 1500 hours. The service life of the lamp is related to the use time and opening frequency of the detector. Therefore, it is necessary to save unnecessary start-up time and reduce the switching frequency to prolong the service life of the xenon lamp during use. If it is accurately judged that the xenon lamp is no longer lit or the energy is too low, a new xenon lamp needs to be replaced. When purchasing a xenon lamp, you should pay attention to the consultation and check whether the model of the xenon lamp matches the model of the instrument. When replacing the xenon lamp, refer to the relevant contents in the instrument manual, paying particular attention to the position sequence of the xenon lamp connection line.
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