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Liquid chromatography column installation and use

Time:2018/09/20   Pageviews:1    Share:
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The LC columnconsists 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.

The LC column 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 LC 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, use a Φ1.57mm connecting tube to insert the hollow screw into the ring, and then tighten the hollow screw. 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 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 silica gel surface bonding - a functional group such as CN, -NH2, so-called bonded phase silica gel.

Reversed-phase column: A non-polar filler which 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 reverse phase packings are also bonded 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 plug from the joints at both ends of the column and put it 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. Always try to reduce the dead volume outside the column when taking over. After the connecting tube passes through the hollow screw and the pressure ring, insert it as far as possible into the end, then tighten the hollow 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 the column is used, the performance of the column is tested and the results are saved 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 condition is that the condition is good.) Only in this way, the measured results are comparable.

1. Pretreatment of the sample:

a, Zui is good to use the mobile phase to dissolve the sample.

b. Use a pretreatment column to remove strong polarities in the sample or impurities that irreversibly adsorb with the column packing.

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 to the 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, the Zui is preferably formulated with a solvent that has a lower UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise bubbles will be easily generated, which may 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 a specific column, to pursue the Zui Jia efficiency, Zui good use zui good 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 zui flow rate is selected, the analysis time may be prolonged. A method of changing the washing strength of the mobile phase can be employed to shorten the analysis time (for example, when a reverse phase column is used, the content of methanol or acetonitrile can be appropriately increased).
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.2ml LC-MS Vials 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, use a Φ1.57mm connecting tube to insert the hollow screw into the ring, and then tighten the hollow screw. 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 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 silica gel surface bonding - a functional group such as CN, -NH2, so-called bonded phase silica gel.
Reversed-phase column: A non-polar filler which 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 reverse phase packings are also bonded 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 plug from the joints at both ends of the column and put it back in the box for spare.

c. According to the flow direction indicated on the column tube, connect 2ml LC-MS Vials 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 with 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. Always try to reduce the dead volume outside the column when taking over. After the connecting tube passes through the hollow screw and the pressure ring, insert it as far as possible into the end, then tighten the hollow 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 the column is used, the performance of the column is tested and the results are saved 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 condition is that the condition is good.) Only in this way, the measured results are comparable.

1. Pretreatment of the sample:

a, Zui is good to use the mobile phase to dissolve the sample.

b. Use a pretreatment column to remove strong polarities 2ml LC-MS Vials in the sample or impurities that irreversibly adsorb with the column packing.

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 to the 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 2ml LC-MS Vials 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, the Zui is preferably formulated with a solvent that has a lower UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise bubbles will be easily generated, which may 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 a specific column, to pursue the Zui Jia efficiency, Zui good use zui good 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 LC column zui flow rate is selected, the analysis time may be prolonged. A method of changing the washing strength of the mobile phase can be employed to shorten the analysis time (for example, when a reverse phase column is used, the content of methanol or acetonitrile can be appropriately increased). 2ml LC-MS Vials

This is the end of the introduction of Liquid chromatography column installation and use. I hope it can help you.

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