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.
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 pipe, 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 pipe (the length of the tube exposed after the connecting pipe passes the compression ring should be strictly controlled at 2.5 mm long or other fixed size).
In the column joints at both ends, a stainless steel filter (or screen) is placed at each end of the column tube to seal the column packing from being lost by the mobile phase being washed 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 3-10 µm. 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.
Second, the use of liquid chromatography column:
Before using the 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 column performance may be different due to the differences in the samples, mobile phase, column temperature and other conditions used; in addition, the column performance test is performed according to the conditions in the chromatographic column factory report (used in the factory test The conditions 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 ability 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 cause chemical reaction with the sample (except for 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 a 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).
note:
1. Because methanol is cheap, the methanol system is recommended for reversed-phase columns (except where acetonitrile must be used).
2. For normal phase columns, it is recommended to use petroleum ether with a boiling range of 30-60 ° C or purified hexane as the mobile phase. Unpurified hexane should not be used. It is best to use ultrapure water (resistivity greater than 18 megohms). Deionized water and double distilled water contain phenolic impurities, which may affect the analysis results.
3. The aqueous mobile phase should be prepared before the experiment, especially in summer when using buffer solution as the mobile phase should not be overnight. It is best to add sodium azide to prevent bacterial growth.
4. The mobile phase requires a 0.45 µm filter membrane to remove particulate impurities.
5. Use HPLC-grade solvents to prepare mobile phases. Proper mobile phases can extend the life of the column and improve column performance.
4. Column performance test:
——Start the liquid chromatograph: a. The flow rate of the mobile phase is set to 1ml / min.
b. The UV detector wavelength is set to 254nm.
——Use the mobile phase composition and test samples used in the factory test.
——Record and calculate the test results.
* Reference (standard JB5226-91) standard chromatographic column for liquid chromatograph test.
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 pipe, 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 pipe (the length of the tube exposed after the connecting pipe passes the compression ring should be strictly controlled at 2.5 mm long or other fixed size).
In the column joints at both ends, a stainless steel filter (or screen) is placed at each end of the column tube to seal the column packing from being lost by the mobile phase being washed 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 3-10 µm. 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.
Second, the use of liquid chromatography column:
Before using the 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 column performance may be different due to the differences in the samples, mobile phase, column temperature and other conditions used; in addition, the column performance test is performed according to the conditions in the chromatographic column factory report (used in the factory test The conditions 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 ability 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 cause chemical reaction with the sample (except for 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 a 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).
note:
1. Because methanol is cheap, the methanol system is recommended for reversed-phase columns (except where acetonitrile must be used).
2. For normal phase columns, it is recommended to use petroleum ether with a boiling range of 30-60 ° C or purified hexane as the mobile phase. Unpurified hexane should not be used. It is best to use ultrapure water (resistivity greater than 18 megohms). Deionized water and double distilled water contain phenolic impurities, which may affect the analysis results.
3. The aqueous mobile phase should be prepared before the experiment, especially in summer when using buffer solution as the mobile phase should not be overnight. It is best to add sodium azide to prevent bacterial growth.
4. The mobile phase requires a 0.45 µm filter membrane to remove particulate impurities.
5. Use HPLC-grade solvents to prepare mobile phases. Proper mobile phases can extend the life of the column and improve column performance.
4. Column performance test:
——Start the liquid chromatograph: a. The flow rate of the mobile phase is set to 1ml / min.
b. The UV detector wavelength is set to 254nm.
——Use the mobile phase composition and test samples used in the factory test.
——Record and calculate the test results.
* Reference (standard JB5226-91) standard chromatographic column for liquid chromatograph test.
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