The liquid chromatograph consists of a high-pressure liquid pump, a detector and a liquid chromatography column. The correct installation and use of the liquid chromatography column is the key to the liquid chromatography work; it is also the correct and reliable liquid chromatography worker. The only way to pass experimental data. First, the installation of liquid chromatography column: 1. Structure of liquid chromatography column: a. The empty column is assembled from column joints, column tubes and filters. The column joints are constructed with a low dead volume. The column joints are threaded assemblies at both ends, with 7/16-inch external threads on one end and 3/16-inch internal threads on the other end (standardized at home and abroad). The 7/16-inch external thread is connected to the 1/4-inch column tube (Φ6.35mm) and placed in the middle for sealing. The 3/16" internal thread is connected to the 1/16" (Φ 1.57mm) connecting tube, and a pressure ring is placed in the middle for the seal of the column joint. In order to minimize the dead volume outside the column, when installing the column, the Φ1.57mm connecting tube should be inserted into the bottom by the hollow screw, and then the hollow screw should be tightened. The pressure ring is pressed and deformed by the hollow screw and then hung on the connecting pipe (the length of the pipe exposed after the connecting pipe passes through the pressure ring should be strictly controlled to 2.5 mm long or other fixed size). In the column joints at both ends, a stainless steel filter (or strainer) is placed at each end of the column tube to block the column packing from being washed out of the column by the mobile phase. Each component of the empty column is made of 316# stainless steel, which can withstand the general solvent effect. However, due to the corrosive nature of the chloride-containing solvent, it should be noted that the solvent and the connecting tube cannot be stored for a long time to avoid corrosion. b, column packing: The separation of the liquid chromatography column is carried out between the filler and the mobile phase, and the classification of the column depends on the type of filler. Normal phase column: mostly silica gel as column packing. According to the appearance, it can be divided into two types: amorphous and spherical. The particle diameter is 3-10. Within the range of μm. Another type of normal phase filler is silica gel surface bonding - a functional group such as CN, -NH2, so-called bonded phase silica gel. Reversed-phase column: A non-polar filler that is mainly based on silica gel and has an octadecyl functional group (ODS) bonded to its surface. There are also amorphous and spherical types. Other commonly used reversed phase fillers are also bonded to C8, C4, C2, phenyl, etc., and the particle size is between 3 and 10 μm. 2, the installation of the column: a. Unpack the column and confirm the type, size, date of manufacture, and solvent stored in the column. b. Unscrew the sealing plugs at the ends of the column and put them back in the box for spare. c. According to the flow direction indicated on the column tube, connect the inlet end of the column to the inlet of the injection valve through the connecting pipe (if the conditions permit, it is recommended to use the guard column in front of the column); the outlet of the column is connected to the detector. The connecting pipe is a stainless steel pipe having an outer diameter of 1.57 mm and an inner diameter of 0.1 to 0.3 mm. Both ends of the connecting tube have a hollow screw and a sealing ring for sealing. When taking over, be sure to try to reduce the dead volume outside the column. After connecting the tube through the hollow screw and the pressure ring, insert it as far as possible into the end, then tighten the cannulated screw clockwise until it stops, then use the wrench to continue to screw 1/4-1/2 turn clockwise, remember not to use too much force. If the column is leaking after being pressurized by the mobile phase, use a wrench to continue to turn 1/4 turn clockwise until it does not leak. Second, the use of liquid chromatography column: Before using the column, it is best to test the performance of the column and save the results as a reference for future evaluation of column performance changes. However, it should be noted that the column performance may vary depending on the conditions of the sample, mobile phase, column temperature, etc.; in addition, the column performance test is performed according to the conditions in the column factory report (factory test) The conditions are the best conditions), and only then, the measured results are comparable. 1. Pretreatment of the sample: a. It is best to use a mobile phase to dissolve the sample. b. Use a pretreatment column to remove strong polarities in the sample or to produce irreversible adsorption impurities with the column packing. c. The particulate impurities were removed by filtration using a 0.45 μm filter membrane. 2. Preparation of mobile phase: Liquid chromatography is the separation of sample components between the column packing and the mobile phase for mass exchange. Therefore, the mobile phase is required to have the following characteristics: a, the flow relative sample has a certain solubility, to ensure that the sample components will not precipitate in the column (or remain in the column for a long time). b. The mobile phase is inert and does not react chemically with the sample (except in special cases). c. The viscosity of the mobile phase should be as small as possible so that a good separation effect can be obtained when using a longer analytical column; at the same time, the column pressure drop is reduced and the life of the liquid pump is prolonged (the temperature can be lowered to reduce the viscosity of the mobile phase). ). d. The physical and chemical properties of the mobile phase should be compatible with the detector used. If a UV detector is used, it is best to use a solvent with a lower UV absorption. e, the boiling point of the mobile phase should not be too low, otherwise it will easily generate bubbles, which will make the experiment impossible. f. After the mobile phase is prepared, it must be degassed. Removal of trace gases dissolved in the mobile phase facilitates both detection and prevention of trace oxygen in the mobile phase from interacting with the sample. 3. Selection of mobile phase flow rate: Since the column efficiency is a function of the linear flow rate of the mobile phase in the column, different column rates can be obtained using different flow rates. For the best column performance for a particular column, it is best to use the optimum flow rate. For a column with an inner diameter of 4.6 mm, the flow rate is generally selected to be 1 ml/min, and for a column having an inner diameter of 4.0 mm, a flow rate of 0.8 ml/min is preferred. When the optimum flow rate is selected, the analysis time may be extended. A method of changing the washing strength of the mobile phase can be employed to shorten the analysis time (for example, when using a reverse phase column, the content of methanol or acetonitrile can be appropriately increased). note: a. Because of the low cost of methanol, it is recommended to use a methanol system for the reversed phase column (except where acetonitrile must be used). b. For the normal phase column, it is recommended to use petroleum ether with boiling range of 30-60 ° C or purified hexane as mobile phase. Unpurified hexane should not be used. It is best to use ultrapure water (resistivity greater than 18 megohms) for water, and phenolic impurities in deionized water and double distilled water, which may affect the analysis results. c. The aqueous mobile phase is most prepared prior to the experiment, especially in summer when the buffer solution is used as the mobile phase. It is best to add sodium azide to prevent bacterial growth. d. The mobile phase is filtered using a 0.45 μm filter to remove particulate impurities. e. Prepare the mobile phase using HPLC grade solvents. Use a suitable mobile phase to extend column life and improve column performance. 4, column performance test: Start the liquid chromatograph: a, the mobile phase flow rate is set to 1 ml / min. b. The UV detector wavelength is set to 254 nm. Use the mobile phase composition and test samples used in the factory test. Record and calculate test results. 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