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 right The only way for experimental data. Second, the use of liquid chromatography column: Liyang Paper Products Co., Ltd. was established in 1999,a professional enterprise integrated in R&D,Marketing and Manufacturing.Liyang Printing provide best solutions and service to various fields of industry.For example,we can make nice and luxury round boxes for products packaging.Such as jewelry Round Box,round box with lid,Cardboard Round Box,etc.Our round boxes with high quality are exquisite packaging for your products.
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1. Installation of liquid chromatography column:
1. Structure of liquid chromatography column:
a. The empty column is assembled by the column joint, column tube and filter.
The column joint adopts a low dead volume structure. The column joint is a threaded component at both ends, one end is 7/16 inch external thread, and the other end is 3/16 inch internal thread (standardized at home and abroad). The 7 / 16-inch external thread is connected to the 1 / 4-inch column tube (Φ6.35mm), and the middle is crushed for sealing. The 3 / 16-inch internal thread is connected to the 1 / 16-inch (Φ1.57mm) connecting tube, and a pressure ring is also placed in the middle for the sealing of the column joint. In order to minimize the dead volume outside the column, when installing the chromatographic column, use the Φ1.57mm connecting tube through the hollow screw to press the ring as far as possible, and then tighten the hollow screw. The compression ring is squeezed and deformed by the hollow screw and tightly clamped on the connecting tube (the length of the tube exposed after the connection tube passes through the compression 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 filter screen) is placed at each end of the column tube to block the column packing from being lost by the mobile phase washing out of the column. The components of the empty column are made of 316 # stainless steel, which can withstand the general solvent effect. However, due to the corrosiveness of the solvent containing chlorides, it should be noted that such solvents should not be left in the column and the connecting tube for a long time to avoid corrosion.
b. Column packing:
The separation of the liquid chromatography column is carried out between the packing and the mobile phase, and the classification of the column depends on the packing type.
Normal phase column: mostly silica gel as the column packing. According to the appearance, it can be divided into two types: amorphous and spherical, and its particle diameter is in the range of 3-10µm. Another type of normal-phase filler is the surface-bonded silica gel-CN, -NH2 and other functional groups, so-called bonded phase silica gel.
Reverse phase column: It is mainly a non-polar packing with silica gel as the matrix and octadecyl functional group (ODS) bonded to its surface. There are also stereotypes and spherical shapes.
Other commonly used reverse phase fillers are bonded C8, C4, C2, phenyl, etc., and their particle size is between 3-10 µm.
2. Installation of chromatographic column:
a. Unpack the column packing box, and confirm the type, size, ex-factory date, and the solvent stored in the column.
b. Unscrew the sealing plugs at the two ends of the column and put them back in the packing box for future use.
c. Connect the inlet end of the chromatographic column to the outlet of the injection valve through the connecting tube according to the flow direction marked on the column tube (if conditions permit, it is recommended to use a guard column before the column); the outlet of the column is connected to the detector. The connecting tube is a stainless steel tube with an outer diameter of 1.57 mm and an inner diameter of 0.1-0.3 mm. Both ends of the connecting tube are provided with hollow screws and sealing pressure rings. When taking over, be sure to try to reduce the dead volume outside the column. Connect the connecting tube through the hollow screw and the pressure ring as far as possible, then tighten the hollow screw clockwise until it can't be tightened, and then continue to turn clockwise 1 / 4-1 / 2 times with a wrench, remember not to use excessive force. If there is liquid leakage after the column is pressurized by the mobile phase, please continue to turn 1/4 turn clockwise with a wrench until there is no liquid leakage.
Before using a liquid chromatography column, it is best to perform column performance tests and save the results as a reference for future evaluation of column performance changes. However, it should be noted that the performance of the liquid chromatography column may be different due to the differences in the conditions of the sample, mobile phase, and column temperature used. In addition, the column performance test is performed in accordance with the conditions in the column factory report ( The conditions used in the factory test are the best conditions), only in this way, the measured results are comparable.
1. Sample pretreatment:
a. It is best to use the mobile phase to dissolve the sample.
b. Use a pre-treatment column to remove impurities that are strongly polarized in the sample or produce irreversible adsorption with the column packing.
c. Use 0.45µm filter membrane to remove particulate impurities.
2. Preparation of mobile phase:
Liquid chromatography is the mass exchange of sample components between the column packing and the mobile phase to achieve the purpose of separation, so the mobile phase is required to have the following characteristics:
a. The mobile phase has a certain dissolving capacity 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 with the sample (except in special cases).
c. The viscosity of the mobile phase should be as small as possible in order to obtain a good separation effect when using a longer analytical column; at the same time, reduce the pressure drop of the column and extend the service life of the liquid pump (the temperature can be reduced by using the method of increasing the temperature ).
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 low UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise bubbles will easily be generated, 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 is not only conducive to detection, but also prevents 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 efficiencies can be obtained using different flow rates. For a particular column, to pursue the best efficiency, it is best to use the best flow rate. For a column with an inner diameter of 4.6mm, the flow rate is generally 1ml / min. For a column with an inner diameter of 4.0mm, a flow rate of 0.8ml / min is preferred.
When the optimal flow rate is selected, the analysis time may be extended. The method of changing the washing intensity of the mobile phase can be used to shorten the analysis time (for example, when using a reversed-phase column, the content of methanol or acetonitrile can be appropriately increased).
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