HAWACH To Talk About The 30 FAQs About HPLC Column (Chapter 1)

High-performance liquid chromatography (HPLC) method has become an important separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commodity inspection, and legal inspection.

HAWACH HPLC columns include the Xchroma series and the Echroma series. Due to the use of different polar modification techniques, the polarities of the stationary phases are significantly different and are widely used for the separation of various hydrophilic and polar compounds. Xchroma series columns use unique polar modification technology, which can provide selectivity different from conventional C18 columns, especially for the separation of compounds with very similar chemical structure, which has its unique advantages.

In view of the increasingly widespread use of HPLC in the entire industry, every laboratory analyst should be proficient in applying HPLC and should be able to analyze some common faults in order to solve problems and improve work efficiency even when running faults. Based on years of experience, HAWACH has summarized 30 FAQs about the HPLC column and this we will talk about the first 5.

Q1: What are the reasons for the retention time drifting or changing rapidly?
On the issue of drift:
①The temperature control is not good: the solution is to use a constant temperature device to keep the column temperature constant;
②The mobile phase changes and we should prevent it from evaporation, reaction, etc.;
③The column is not well balanced, and the column needs to be balanced for a longer time.

High Quality HPLC Columns

About rapid changes
①About the change of flow rate, the operator needs to reset the flow rate to keep it stable;
②There are air bubbles in the pump, which can be expelled by exhausting and other operations;
③The mobile phase is not suitable. The solution is to change the mobile phase or mix the mobile phase in the control room.

Q2: What are the reasons for tailing or double peaks?
①The sieve plate is blocked or the column is invalid: the solution is to reversely wash the column, replace the sieve plate or replace the column;
②There are interference peaks. To solve it, the operator needs to use a longer column; change mobile phase or replace the column with good selectivity;
③The column may be overloaded and reduce the injection volume.

Q3: What are the main reasons and solutions for insufficient HPLC sensitivity?
① The sample volume is insufficient: the solution is to increase the sample volume;
② The sample did not flow out of the column. The mobile phase or column can be changed according to the chemical nature of the sample;
③ The sample does not match the detector. Adjust the wavelength or change the detector according to the chemical nature of the sample;
④ Too much attenuation of the detector. Just adjust the attenuation;
⑤ The time constant of the detector is too large. The solution is to reduce the time parameter;
⑥ Pollution of the detector pool window. The solution is to clean the pool window;
⑦ There are bubbles in the detection pool. The solution is to exhaust air;
⑧ The pressure measuring range of the recorder is inappropriate. Just adjust the voltage range;
⑨ Mobile phase flow is not suitable. Just adjust the flow rate;
⑩ Detector and recorder exceed the calibration curve. The solution is to check the recorder and detector and make the calibration curve again.

Q4: When doing HPLC analysis, the column pressure is unstable, what is the reason? How to solve it?
① Proportional valve fails: just replace the proportional valve;
② The pump seal is damaged: just replace the seal;
③ The bubbles in the solvent: the solution is to degas the solvent, and change the degassing method if necessary;

④ Check the leaks systematically, find the leaks and seal them;
⑤ Gradient elution, pressure fluctuation is normal at this time.

Q5: The operator replaced another grade of ODS column, but the retention time cannot be reproduced, why?
This is because the analyte may have the ability to form hydrogen bonds. Although the manufacturing technology of fillers has greatly improved in the past few years, the concentration of silanol groups on the surface of ODS fillers of different manufacturers is different. It is these silanol groups that may interact with the sample.

Therefore, the relative retention times of the components of the same analyte on different ODS columns may be different. Adding a small number of competitors in the mobile phase, such as triethylamine (TEA), will saturate the bonding ability of the silanol group, thus ensuring that the relative retention time on different grade columns has good reproducibility. If the separation situation is ok, the system is stable and the system suitability requirements are met, so there is no need to reproduce the retention time.
(To be continued)