What issues should be considered when using liquid chromatography?
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What issues should be considered when using liquid chromatography?
1. The sample solution contains no mechanical impurities； 2. Manual sampling: Thoroughly clean the syringe, effectively remove air bubbles, ensure accurate quantification; the sampling valve should switch quickly, and after sampling, clean the valve with ultrapure water ； 3. Automatic sampling: Ensure an adequate depth of the sample solution (to prevent the needle tip from not being submerged below the liquid surface) ； Ensure that the sample solution is free of bubbles, especially when using a cannula ； The bottle cap should be tight enough; if it’s too loose, the solvent in the sample will evaporate, and if it’s too tight, the bottle gasket may deform and fall off ； Use bottle pads that are not too old, to prevent debris from clogging the needle holes when piercing.
1. The issue of the mobile phase in the chromatography column being depleted: Many people who conduct chromatographic separation experiments have encountered this situation: they fail to replenish the mobile phase in time, and the pump draws all the liquid from the solvent bottle, causing the HPLC system to stop working. Can this situation damage the chromatography column? Does the pump drain all the mobile phase from the column? Can the column still be used? In fact, if the pump drains the liquid from the solvent bottle, it does not cause any damage to the chromatography column. Even if the pump is filled with air, it cannot push air into the column, as pumps are designed to transport only liquids, not air.
2. The issue of the chromatography column drying out: Another possible scenario is that the sealing caps at both ends of the column are forgotten to be closed, or they are not sealed properly, resulting in the column drying out. Again, it’s unlikely for the entire column to dry out; it’s more probable that only a few millimeters at each end of the column dry out, as it takes a considerable amount of time for all the solvent to evaporate. Even if the column does dry out, it isn’t necessarily beyond repair. One can try flushing the column with a completely degassed solvent with low surface tension (such as methanol degassed with helium) to remove any gases present. A lower surface tension helps to wet the surface of the packing material； The degassed solvent should be able to dissolve and remove the gases trapped in the packing. It takes about an hour or more to thoroughly saturate the chromatography column (at a flow rate of 1 mL/min) so that it can return to its normal state. 3. When using PEEK (polyetheretherketone) tubing and fittings, the following points need to be taken into account: If it is necessary to frequently change the flow path or replace chromatography columns of different brands, tubing and fittings made of PEEK are very convenient to use. PEEK tubing is easy to connect ； PEEK connectors not only can be secured by hand tightening without the need for tools, but it is also easy to adjust the length of the tubing outside the cone-shaped clamp, which facilitates connection to chromatography columns of different brands or specifications. When using tubing made of this material, it should be noted that PEEK has poor compatibility with halogenated hydrocarbons and furan. Although no obvious signs of these solvents dissolving the PEEK material have been observed, PEEK becomes brittle when exposed to them. Another factor to consider is the pressure limit: stainless steel tubing can withstand pressures of up to 6000 psi, while PEEK tubing can only handle around 4000 psi (although the pressure in most HPLC systems does not exceed 3000 psi). When using PEEK connectors, there is no need to worry about their solvent resistance, as the connectors almost never come into direct contact with solvents. However, the pressure limit of hand-tightened PEEK connectors is lower than that of stainless steel tubing; therefore, high pressures may cause the connectors to slide within the tubing, resulting in dead volume or leakage. 4. Preventing failures in liquid phase pumps: A pump that is in good working condition should enable a stable baseline on the chromatogram ； The retention time exhibits good repeatability. The pressure fluctuation is less than 2% during isocratic elution. During gradient elution, the pressure changes should be slow and gradual. To maintain the proper functioning of the pump, it is necessary to keep the system clean, ensure the quality of solvents and reagents, and filter as well as degas the mobile phase. The following are some measures to prevent pump failures: 1) Use high-quality reagents and HPLC-grade solvents ； 2) Filter the mobile phase and solvent ； 3) Degassing ； 4) Empty the exhaust gas at the start of use each day, and wash away the buffer from the pump after operation is completed ； 5) Prevent water or corrosive solvents from remaining in the pump ； 6) Regularly replace the washers ； 7) Add lubricant as needed ； 8) Consult other recommendations in the pump operation manual. 9) To resolve faults as quickly as possible, it is advisable to keep on hand components such as seals, check valves (for inlet and outlet), pump heads, various types of connectors, fuses, as well as replacement tools.
2. The issue of the chromatography column drying out: Another possible scenario is that the sealing caps at both ends of the column are forgotten to be closed, or they are not sealed properly, resulting in the column drying out. Again, it’s unlikely for the entire column to dry out; it’s more probable that only a few millimeters at each end of the column dry out, as it takes a considerable amount of time for all the solvent to evaporate. Even if the column does dry out, it isn’t necessarily beyond repair. One can try flushing the column with a completely degassed solvent with low surface tension (such as methanol degassed with helium) to remove any gases present. A lower surface tension helps to wet the surface of the packing material； The degassed solvent should be able to dissolve and remove the gases trapped in the packing. It takes about an hour or more to thoroughly saturate the chromatography column (at a flow rate of 1 mL/min) so that it can return to its normal state. 3. When using PEEK (polyetheretherketone) tubing and fittings, the following points need to be taken into account: If it is necessary to frequently change the flow path or replace chromatography columns of different brands, tubing and fittings made of PEEK are very convenient to use. PEEK tubing is easy to connect ； PEEK connectors not only can be secured by hand tightening without the need for tools, but it is also easy to adjust the length of the tubing outside the cone-shaped clamp, which facilitates connection to chromatography columns of different brands or specifications. When using tubing made of this material, it should be noted that PEEK has poor compatibility with halogenated hydrocarbons and furan. Although no obvious signs of these solvents dissolving the PEEK material have been observed, PEEK becomes brittle when exposed to them. Another factor to consider is the pressure limit: stainless steel tubing can withstand pressures of up to 6000 psi, while PEEK tubing can only handle around 4000 psi (although the pressure in most HPLC systems does not exceed 3000 psi). When using PEEK connectors, there is no need to worry about their solvent resistance, as the connectors almost never come into direct contact with solvents. However, the pressure limit of hand-tightened PEEK connectors is lower than that of stainless steel tubing; therefore, high pressures may cause the connectors to slide within the tubing, resulting in dead volume or leakage. 4. Preventing failures in liquid phase pumps: A pump that is in good working condition should enable a stable baseline on the chromatogram ； The retention time exhibits good repeatability. The pressure fluctuation is less than 2% during isocratic elution. During gradient elution, the pressure changes should be slow and gradual. To maintain the proper functioning of the pump, it is necessary to keep the system clean, ensure the quality of solvents and reagents, and filter as well as degas the mobile phase. The following are some measures to prevent pump failures: 1) Use high-quality reagents and HPLC-grade solvents ； 2) Filter the mobile phase and solvent ； 3) Degassing ； 4) Empty the exhaust gas at the start of use each day, and wash away the buffer from the pump after operation is completed ； 5) Prevent water or corrosive solvents from remaining in the pump ； 6) Regularly replace the washers ； 7) Add lubricant as needed ； 8) Consult other recommendations in the pump operation manual. 9) To resolve faults as quickly as possible, it is advisable to keep on hand components such as seals, check valves (for inlet and outlet), pump heads, various types of connectors, fuses, as well as replacement tools.
Where are the engineering companies that specialize in chromatographic separation mainly located? Is it common in Jiangsu, Zhejiang, and Shanghai?