HAWACH to Talk About the 30 FAQs About HPLC Column (Chapter 2 Of Q6-Q10)

Q6: Why is the column pressure too high during the HPLC column acceptance test?
Too high column pressure is a problem often encountered by HPLC column users. There are many reasons, and it is often not the problem of the pillar itself. You can check the cause of the problem by following the steps below.
① Remove the protection column to see if the HPLC column pressure is still high, otherwise, it is a problem to the protection column.
② Remove the HPLC chromatographic column from the instrument to check whether the pressure drops. If not, the pipeline is blocked and needs to be cleaned. If yes, check again;
③ Connect the inlet and outlet of the column to the instrument in reverse and rinse the column with 10 times the volume of the mobile phase (do not connect the detector at this time to prevent solid particles from entering the flow cell). At this time, if the column pressure still does not drop, check again, which is only for the used column.
④ Replace the sieve plate at the HPLC column entrance. If the column pressure drops, it means the solvent or sample contains particulate impurities. It is these impurities that block the sieve plate and cause the pressure to rise. If the column pressure is still high, please contact the manufacturer. Under normal circumstances, an online filter can be connected between the injector and the guard column to avoid the problem of excessive column pressure.

Q7: Will the mobile phase in the column drain?
Many people doing chromatographic separation experiments have encountered such a situation that the mobile phase in the solvent bottle is sucked dry by accident and the mobile phase in the solvent bottle is inadvertently replenished, and the HPLC system stops working. Will this damage the column? Has the pump drained all mobile phases from the HPLC column? Can the HPLC column be used? In fact, if the pump dries the mobile phase, it will not cause damage to the column. Even if the pump is filled with air, the pump will not exhaust the air into the HPLC column. Because the pump can only deliver liquid, not air.

In contrast, another situation that is more likely to happen is to forget to close the sealing caps on both ends of the column or the cap is too loose and the HPLC column dries. Similarly, it is not easy for the entire column to dry up. It is possible that only a few millimeters at both ends of the column have dried out. It takes a long time for the column to dry out because all the solvent has evaporated.

You can try to flush the column with a completely degassed solvent with low surface tension (such as methanol degassed with helium) to remove the air. Lower surface tension helps to wet the surface of the filler; the degassed solvent should be able to dissolve and remove the air trapped in the filler. The HPLC column takes approximately one hour or more (at a flow rate of 1 ml /min) to be thoroughly infiltrated and returned to be normal.

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Q8: What are the causes and solutions of baseline drift?
1. Cause analysis
① Column temperature fluctuates. Even a small temperature change will cause the baseline to fluctuate. It usually affects differential detectors, conductivity detectors, lower sensitivity UV detectors, or other photoelectric detectors.
② The mobile phase is not uniform.
③ The flow cell has air or is contaminated.
④ The detector outlet is blocked. The high pressure causes the flow cell window to rupture, producing a noise baseline.
⑤ Improper matching of mobile phase or change of flow rate.
⑥ Slow column equilibrium, especially when the mobile phase changes.
⑦ Mobile phase pollution, deterioration, or made up of low-quality solvents.
⑧ Substances with strong retention (high K’ value) in the sample. The sample is eluted, thus showing a gradually rising baseline.
⑨ The use of recycled solvents is not recommended. The detector is not adjusted.
⑩ The detector is not set at the maximum absorption wavelength.

① Control the temperature of the column and mobile phase, and use a heat exchanger before the detector.
② Use HPLC grade solvent, high purity salt, and additives. The mobile phase is degassed before use, and online degassing or helium degassing is used during use.
③ Flush the flow cell with methanol or other strong polar solvents. If necessary, 1N nitric acid can be used. (Do not use hydrochloric acid).
④ Remove the obstruction or replace the tube. Replace the flow cell window, according to the User Manual.
⑤ Change the ratio or flow rate. To avoid this problem, periodically check the mobile phase composition and flow rate.
⑥ Rinse with a medium-strength solvent. When changing the mobile phase, rinse with 10-20 times the volume of the new mobile relative HPLC column before analysis. When using ion-pair reagents and buffer salts, attention should be paid to the equilibration HPLC column.
⑦ Check the composition of the mobile phase. Use high-quality chemical reagents and HPLC grade solvents to change the analysis conditions. Use guard HPLC columns and, if necessary, periodically flush the HPLC column with a strong solvent between injections or during analysis.
⑨ Reset the baseline. Use a new mobile phase.
⑩ Adjust the wavelength to the maximum absorption wavelength. Reselect the detection wavelength.

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Q9: What are the reasons for regular baseline noise?
① There is air in the mobile phase, detector or pump (sharp peak)
② Leakage.
③ The mobile phase is not completely mixed.
④ Temperature influence (column temperature is too high, the detector is not heated)
⑤ There are other electronic devices on the same line (accidental noise)
⑥ Pump vibration

① Deair mobile phase. Flush the system to remove air from the detector or pump.
② Check whether the pipe joint is loose, whether the pump is leaking, whether there is salt precipitation, and abnormal noise. If necessary, replace the pump seal.
③ Shake by hand to mix evenly or use low viscosity solvent.
④ Reduce the difference or add a heat exchanger.
⑤ Disconnect the LC, detector, and recorder, check whether the interference comes from outside, and correct it. Adopt precision-grade regulated power supply.
⑥ Pulse damper is added to the system.

Q10: What are the reasons for irregular baseline noise?
① Leakage.
② The mobile phase is contaminated, deteriorated, or made up of low-quality solvents.
③ The solvents in the mobile phase are not miscible.
④ There are problems with the electronic components of the detector/recorder
⑤ There are bubbles in the system.
⑥ There are bubbles in the detector.
⑦ Pollution of the flow cell (Even a few pollutants will generate noise.)
⑧ Insufficient energy of the detector lamp.
⑨ Loss or blockage of HPLC chromatographic column packing.
⑩ Uneven mixing of mobile phase or abnormal working of the mixer.

① Check whether the joint is loose, whether the pump is leaking, whether there is salt precipitation, and abnormal noise. If necessary, replace the seal. Check whether the flow cell leaks.
② Check the composition of the mobile phase.
③ Choose mobile phases that are miscible.
④ Disconnect power to the detector and recorder, check, and correct.
⑤ Clean the system with a strong polar solution.
⑥ Clean the detector and install a background pressure regulator behind the detector.
⑦ Clean the flow cell with 1N nitric acid (no phosphoric acid).
⑧ Replace the lamp.
⑨ Replace the HPLC column.
⑩ Repair or replace the mixer. When the mobile phase does not follow the gradient, it is recommended not to use the mixing device of the pump.
(to be continued)