First, the PH of the mobile phase should be within the range of use
Due to damage to the column caused by improper pH control of the mobile phase, it is often difficult to recover the column, so care must be taken to strictly control the pH of the mobile phase.
Second, remove the solid particles in the sample and mobile phase
The solid particulate matter contained in the sample and mobile phase will block the chromatographic sieve plate. Blocking the sieve plate will not only cause the column pressure to rise, but also cause the column efficiency to drop, because the blockage of the sieve plate will cause uneven flow. , causing the chromatographic peak shape to be tailed and widened, thereby reducing the efficiency of the column. Therefore, it is recommended to use ultrapure water and chromatographically pure reagents to filter the sample before the sample is analyzed, and the mobile phase is passed through a 0.45 μm filter.
Third, use the guard column or online filter
After filtration of the sample and mobile phase, the solid particulate matter cannot be completely eliminated, because the wear of the pump, the aging of the seal ring and the pipeline will also produce solid particulate matter, which is carried by the mobile phase into the column and blocks the sieve plate, resulting in The column pressure is increased and the column efficiency is lowered. Both the guard column and the in-line filter have sieve plates with the same pore size as the column pores, thus preventing solid particles from reaching the column and preventing blockage of the column. Since the increase in column pressure is a large percentage of analytical failures, it is recommended that you add a guard column or inline filter to the column injection end, in addition to filtering the sample and mobile phase.
If the column pressure rise is due to plugging of the injection end screen, you can choose the following way to remedy:
1. Add a guard column or an in-line filter in front of the column, and then reverse-collapse the column with methanol and water = 20/80 ml/min for 180 min.
2. Add a guard column or inline filter to the column injection end and use it in reverse.
Fourth, the correct use of buffer salts
The buffer salt is usually soluble in water and is insoluble in organic solvents. Therefore, improper use of the buffer salt causes it to precipitate, blocking the pores between the filler matrix and the voids between the particles, causing the filler to be kneaded and the column pressure to rise; The bonded carbon chain is free to stretch, which reduces the retention capacity of the column and reduces the efficiency of the column. After the buffer salt is precipitated, removal is very difficult, so proper use of buffer salts is very important to extend the life of the column.
5. Prevent strong retained substances from remaining on the column
Strongly retained substances and macromolecular compounds accumulate in the column, creating additional retention behavior for the compounds in the sample, which not only causes peak broadening, tailing, but also decreases column efficiency, and also causes retention time changes, accumulating To a certain extent, it will also cause the column pressure to rise. Since the effect of strongly retained substances and macromolecular compounds on chromatographic separation is a cumulative effect, it takes a certain amount of time to be reflected, but for many drugs, especially complex samples, it is difficult to judge whether they contain strong retained substances, so To prevent the accumulation of strongly retained materials, the column needs to be cleaned with pure methanol or acetonitrile in daily routine maintenance.
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